Systems and methods for supporting multiple automated work-flows

ABSTRACT

Systems and methods for automated workflow comprise assigning a set of first targets to an uncompiled first workflow. The uncompiled first workflow specifies a first set of process modules. Each such module is associated with a subset of unit operations. Each unit operation includes a time interval and specifies an instrument. For each target in the set of first targets, the uncompiled workflow is translated into an instance of a compiled first workflow comprising a linear temporal order of unit operations, each including execution instructions for an addressed instrument. A set of second targets is obtained and assigned a second uncompiled workflow. Compilation of the second uncompiled workflow for each second target produces a different instance of a compiled second workflow. Each second compiled workflow comprises a linear temporal order of unit operations, with each unit operation including execution instructions for an addressed instrument and specifying a time interval.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of InternationalPatent Application No. PCT/US18/14751, entitled “Systems and Methods forSupporting Multiple Automated Work-Flows and Changing Between Them,”filed Jan. 22, 2018, which in turn claims priority to U.S. ProvisionalPatent Application No. 62/448,948, filed on Jan. 20, 2017, entitled“Systems and Methods for Supporting Multiple Automated Work-Flows andChanging Between Them,” each of which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Jan. 22, 2018, isnamed 120568-5001-WO_ST25.txt and is 559 bytes in size.

BACKGROUND Field

The present disclosure relates to systems and methods for supportingbiological foundries. More particularly, the present disclosure relatesto a systems and methods for fully automated workflows using biologicalfoundries.

Description of Related Art

Electronic devices and components (hereinafter “instruments”) have foundnumerous applications in chemistry and biology (more generally, “lifesciences”), especially for detection and measurement of various chemicaland biological reactions and identification, detection and measurementof various compounds, and the synthesis of such compounds, to name a fewapplications. Biological foundries, which comprise lab instruments thatare in electronic communication with each other, are being increasingused to automate and handle these applications. Biological foundries canbe complex and expensive. Moreover, efficient use of such foundriespresents a difficult scheduling problem. For instance, two differentprocesses operating at the foundry may need to use the same instrument.Without some consideration for scheduling, conflicts may arise where twodifferent processes request the same instrument. Moreover, without someconsideration for scheduling, the foundry may be under-utilized, withthe foundry proceeding to process tasks at some form of lowest commondenominator associated with the foundry.

During operation of the foundry, instruments must be preciselycontrolled in order to ensure proper use, while research materialsrequire meticulous care in order to prevent contamination or spillage.Conventionally, a research scientist is responsible for manuallyhandling samples and actively operating instruments. The researchscientist must plan, weigh, and dispense sample batches; sterilize andwash instruments; and then place the sample batches into a variety ofinstruments. Due to human nature, these sample batches are ofteninefficiently planned, incorrectly weighed, contaminated, or dropped;any one of which may impair workflow.

To consider the depth and complexity that foundries are capable ofhandling, consider the uses of the transcription activator-like effectornuclease (TALENs), which is a highly efficient and programmable genomeediting tool that has been applied in a wide range of organisms (Sun etal., 2012, “Recent advances in targeted genome organic engineering inmammalian systems,” Biotechnol J 7 (9), p 1074). A TALEN comprises aFokI DNA cleavage domain and a DNA binding domain (DBD) that has tandemrepeats of a 33-35 amino acids (aa) motif. The twelfth and thirteenthamino acid residue within each repeat is known as repeat-variabledi-residue (RVD), and it determines the DNA binding specificity of therepeat. By assembling repeats with specific RVDs in order, a TALeffector DBD can bind to a specific DNA sequence (Boch, 2011, “TALEs ofgenome targeting,” Nat Biotechnol 29 (2), p 135). Because FokI cleavagedomain functions as a dimer, TALENs are typically used in tail-to-tailheterodimeric pairs to create double stranded breaks for genome editing(Miller et al., 2011, “A TALE nuclease architecture for efficient genomeediting,” Nat Biotechnol 29 (2), p 143). Such heterodimeric designgenerates high editing efficiency and improves specificity, but alsopresents challenges in TALEN synthesis as well as usage. A number ofmethods have been developed to synthesize TALEN expression DNA vectors(Briggs et al., 2012, “Iterative capped assembly: rapid and scalablesynthesis of repeat-module DNA such as TAL effectors from individualmonomers,” Nucleic Acids Res, 40 (15), e117; Reyon et al., 2012, “FLASHassembly of TALENs for high-throughput genome editing,” Nat Biotechnol,30 (5), p 460; Ding et al., 2013, “A TALEN genome-editing system forgenerating human stem cell-based disease models,” Cell Stem Cell, 12(2), p 238; Kim et al., 2013, “A library of TAL effector nucleasesspanning the human genome,” Nat Biotechnol, 31 (3), p 251; Schmid-Burgket al., 2013, “A ligation-independent cloning technique forhigh-throughput assembly of transcription activator-like effectorgenes,” Nat Biotechnol, 31 (1), p 76).

Taking advantage of an optimized set of four base-pair junctions as wellas preassembled di-repeat part library, a one-step assembly scheme wasdeveloped based on the Golden Gate method using a foundry (Liang et al.,2014, “FairyTALE: A high-throughput TAL effector synthesis platform,”ACS Synth Biol, 3 (2), p 67). Custom TALEN vectors could be constructedin 24 hours at 96% success rate and a material cost of five dollars.These methods, however, can only assemble vectors harboring a singleTALE-FokI monomer. Since TALEN requires a heterodimer to make a cut, twomonomers are introduced into the host cells either on two separatevectors or a single sub-cloned vector with both monomers. Either optionhas significant drawbacks. For example, both of them will require twiceas many vectors synthesized as the number of target sequences. When themonomers are on separate vectors, the number of cells transfected ortransformed with both monomers can be reduced. More importantly, thedual vector scheme makes it very difficult to perform high throughputgenetic screening. Thanks to fluorescence-activated cell sorting (FACS)and next-generation sequencing, a large number of cells with differentgenotypes can be screened for phenotypes of interest and sequenced(Shalem et al., 2014, “Genome-scale CRISPR-Cas9 knockout screening inhuman cells,” Science, 343 (6166), p 84; Wang et al., 2014, “GeneticScreens in Human Cells Using the CRISPR-Cas9 System,” Science, 343(6166), p 80). As a precision genome editing tool, TALEN can potentiallybe used to generate a genomic knock-out library. However, because thetwo monomers of each TALEN pair need to be introduced to the same cell,library transfection or transformation is not possible using a dualvector system. Moreover, current methods to construct a single-vectorTALEN require a lengthy and complicated subcloning procedure, whichmakes the synthesis process difficult to scale up. A high-throughputsynthesis method for single-vector TALENs using a foundry will open upnew possibilities.

Thus, prior to the present disclosure there existed a need for fullyautomated platform to custom manufacture TALENs in a versatilebiological foundry. This is just one example of the many needs forimproved biological foundries.

The information disclosed in this Background section is only forenhancement of understanding of the general background of the inventionand should not be taken as an acknowledgement or any form of suggestionthat this information forms the prior art already known to a personskilled in the art.

BRIEF SUMMARY

Advantageously, the systems and methods for supporting fully automatedworkflows detailed in the present disclosure address the shortcomings inthe prior art detailed above.

Transcription activator-like effector nuclease (TALEN) is a programmablegenome editing tool with wide applications. Since TALENs performcleavage of DNA as heterodimers, a pair of TALENs must be synthesizedfor each target genome locus. Conventionally, TALEN pairs are eitherexpressed on separate vectors or synthesized separately and thensubcloned to the same vector. Neither approach allows high-throughputconstruction of TALEN libraries for large-scale applications. Here wepresent a single-step assembly scheme to synthesize and express a pairof TALENs in a single transcript format with the help of a P2Aself-cleavage sequence. Furthermore, we developed a fully automatedplatform to custom manufacture TALENs in a versatile biological foundry.Using the systems and methods of the present disclosure, four hundredpairs of TALENs can be synthesized with over 96.2% success rate at areasonable material cost per pair. This platform opens the door toTALEN-based genome-wide studies, as well as many other applications inthe life sciences.

Building on our previously published “FairyTALE” protocol (Liang et al.,2014, “FairyTALE: A high-throughput TAL effector synthesis platform,”ACS Synth Biol, 3 (2), p 67), we sought to assemble a pair of TALENmonomers onto a single vector in a one-step reaction. In previous work,2A self-cleavage peptide (Donnelly et al., 2004, “Multiple gene productsfrom a single vector: ‘self-cleaving’ 2A peptides,” Gene Ther, 11 (23),p 1673; Kim et al., 2011, “High Cleavage Efficiency of a 2A PeptideDerived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish andMice,” Plos One, 6 (4)) was used to co-transcribe a pair of TALENs asone mRNA molecule but translated as separate functional proteins (Cermaket al., 2015, “High-frequency, precise modification of the tomatogenome,” Genome Biol, 16, p 232; Mariano et al., 2014, “Highly efficientgenome editing via 2A-coupled co-expression of two TALEN monomers. BMCRes Notes, 7, p 628; Xu et al., 2013, “Targeted Myostatin Gene Editingin Multiple Mammalian Species Directed by a Single Pair of TALENucleases,” Mol Ther Nucleic Acids, 2, e112). We operationalized thisco-expression strategy in a 15-insert one-pot assembly scheme, andassembled single-plasmid TALENs in one step at more than 87.7% fidelity.TALENs synthesized using this one-step single-transcript design hadcomparable cleavage activity in mammalian cells as those synthesizedusing a two-plasmid design. We implemented the synthesis on iBioFAB(Illinois Biofoundry for Advanced Biomanufacturing), an integrated andversatile robotic system, to fully automate the synthesis process. Inaccordance with the present disclosure, four hundred pairs of TALENs canbe generated on a daily basis at a material cost of $2.1 per pair withminimal human intervention. We envision that genome-wide studies usingTALENs can be scaled up to screen hundreds of loci in parallel with sucha simplified design and automated synthesis.

Accordingly, various aspects of the present disclosure are directed toproviding systems and methods for supporting multiple automatedworkflows in a biological foundry.

One aspect of the present disclosure provides a non-transitory computerreadable storage medium for implementing a workflow. The non-transitorycomputer readable storage medium stores instructions, which whenexecuted by a first device, cause the first device to obtain a firstplurality of organic engineering targets and assign the first pluralityof organic engineering targets to a first uncompiled workflow. The firstuncompiled workflow is configured to produce the first plurality oforganic engineering targets and is associated with a first subset ofprocess modules in a plurality of process modules. Each respectiveprocess module in the plurality of process modules is associated with adifferent subset of unit operation definitions in a plurality of unitoperation definitions. Each respective unit operation definition in theplurality of unit operation definitions is independently associated witha corresponding time interval. Each respective unit operation definitionin the plurality of unit operations is independently associated with afirst subset of instruments in a plurality of instruments (e.g.,biofoundry).

The instructions further cause the first device to translate, for eachrespective organic engineering target in the first plurality of organicengineering targets, the first uncompiled workflow into a correspondinginstance of a compiled first workflow for the respective organicengineering target. The corresponding instance of the compiled firstworkflow comprises, for each respective instrument in the first subsetof instruments, an address of the respective instrument and one or moreexecution instructions for the respective instrument, as well as a firstplurality of unit operations. The first plurality of unit operations istemporally organized into a linear temporal order. Each respective unitoperation in the first plurality of unit operations is characterized bythe time interval of the corresponding unit operation definition,thereby forming a plurality of instances of the compiled first workflow.

Additionally, the instructions further cause the first device to obtaina second plurality of organic engineering targets and to assign thesecond plurality of organic engineering targets to a second uncompiledworkflow. The second uncompiled workflow is configured to produce thesecond plurality organic engineering targets and is associated with asecond subset of process modules in the plurality of process modules.The instructions further cause the first device to translate, for eachrespective organic engineering target in the second plurality of organicengineering targets, the second uncompiled workflow into a correspondinginstance of a compiled second workflow for the respective organicengineering target. The corresponding instance of the compiled secondworkflow comprises for each respective instrument in the second subsetof instruments an address of the respective instrument and one or moreexecution instructions for the respective instrument, as well as asecond plurality of unit operations. The second plurality of unitoperations is temporally organized into a linear temporal order. Eachrespective unit operation in the second plurality of unit operations ischaracterized by the time interval of the corresponding unit operationdefinition. A time interval of a unit operation in the second pluralityof unit operations is adjusted from a time interval of the correspondingunit operation definition by an amount in accordance with adetermination of an interlocking condition with a unit operation in thefirst compiled workflow, thereby forming a plurality of instances of thecompiled second workflow.

In some embodiments, the first or second uncompiled workflow is selectedfrom the group consisting of cloning, evolutionary organic engineering,genome organic engineering, genotyping, library screening, pathwayconstruction, and protein organic engineering.

In some embodiments, the plurality of process modules comprises two ormore process modules selected from the set of cell culture, DNAassembly, DNA purification, DNA quantification, normalization,polymerase chain reaction (PCR), protein extraction, sample analysis,sample preparation, sampling, and transformation. In some embodiments,the plurality of process modules comprises three or more process modulesselected from the above set of process modules.

In some embodiments, the plurality of unit operation definitionscomprises two or more unit operation definitions from the set ofcentrifugation, chilled incubation, chromatography, colony selection,colony separation, dispensing, electrophoresis, electroporation, heatedincubation, labelling, magnetic separation, mass spectrometry, peeling,pipetting, plate reading, sealing, shaking incubation,spectrophotometry, and thermo-cycling. In some embodiments, theplurality of unit operation definitions comprises three or more unitoperation definitions from the above set of unit operation definitions.

In some embodiments, the plurality of instruments comprises two or moreinstruments from the set of a liquid handling robot, a temperaturecontrolled block, a microplate reader, a chilled incubator, a heatedincubator, a shaking incubator, a reagent dispenser, a plate centrifuge,a storage carousel, a de-lidding station, a blow-dryer, a plate sealer,a label printer, a pipetting device, a shaker, a light box, and acamera. In some embodiments, the plurality of instruments comprisesthree or more instruments from the above set of instruments. In someembodiments, the plurality of instruments comprises four or moreinstruments from the above set of instruments. In some embodiments, theplurality of instruments comprises five or more instruments from theabove set of instruments.

In some embodiments, the address of the respective instrument comprisesCartesian coordinates, polar coordinates, spherical coordinates, jointcoordinates, or tool coordinates of the respective instrument. In someembodiments, the address of the respective instrument comprises aphysical location of the respective instrument. In some embodiments, theaddress of the respective instrument comprises a unique electronicaddress of the respective instrument.

In some embodiments, the corresponding instance of the respectivecompiled workflow further comprises an operating condition for therespective instruction.

In some embodiments, the non-transitory computer readable storage mediumfurther stores instructions for enabling a user of the first device,(e.g., via a graphical user interface), to adjust the linear temporalorder of the first plurality of unit operations. In some embodiments,the non-transitory computer readable storage medium further storesinstructions for enabling a user of the first device to adjust thelinear temporal order of the first plurality of unit operations withoutusing graphical user interface.

In some embodiments, the translating further comprises validating thesecond plurality of unit operations according to a predeterminedvalidation list. The predetermined validation list comprises one or morecriteria of the compiled second workflow. In some embodiments, the oneor more criteria of the compiled second workflow comprises a priority ofeach unit operation in the second plurality of unit operations, a weightof each unit operation in the second plurality of unit operations, atime of completion for the second plurality of unit operations, acompatibility of the second plurality of unit operations to a differentplurality of unit operations, a property of each unit operation in thesecond plurality of unit operations, and one or more constraints of thesecond plurality of unit operations.

In some embodiments, the property of each unit operation in the secondplurality of unit operations is selected from the set of a viscosityvalue, a purity value, a composition value, a temperature value, aweight value, a mass value, and a volume value.

In some embodiments, the first device is in electronic communicationwith at least one transport path coupled to the plurality of instrumentsfor receiving a sample from the plurality of instruments and returningthe sample to the plurality of instruments. In some embodiments, thetransport path comprises at least one transporter configured to moveabout the transport path, and a physical storage medium disposed on theat least one transporter. In some embodiments, the at least onetransporter comprises a robotic arm, a ground vehicle, a drone, aconveyor belt, a transfer station, a lift, a crane, an elevator or acombination thereof. In some embodiments, the at least one transporterfurther comprises a liquid handling robot.

In some embodiments, the second plurality of organic engineering targetsare determined from outputs of the plurality of instances of thecompiled first workflow.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets is an input into acorresponding instance of a compiled first workflow in the plurality ofinstances of the compiled first workflow.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets is an output of a correspondinginstance of a compiled first workflow in the plurality of instances ofthe compiled first workflow.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets is an assembly of nucleic acidcomponents.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets is a plurality of reagents ofnucleic acid components.

In some embodiments, each respective compiled workflow in the pluralityof instances of the compiled first workflow is a scheme to synthesizeand express a pair of TALENs in a single transcript format by a P2Aself-cleavage sequence. In some embodiments, at least 400 pairs ofTALENs are expressed in a 24-hour time interval.

In some embodiments, the instructions, when executed by the firstdevice, further causes the first device to export the output of acorresponding instance of a compiled first workflow to a second device.

In some embodiments, the first device communicates with at least oneexternal control server or an external database server.

In some embodiments, the instructions, when executed by the firstdevice, further causes the first device to save workflow data describingdata of the executed instructions.

In some embodiments, the non-transitory computer readable storage mediumfurther comprises instructions for concurrently executing one or moreinstances of the compiled first workflow and one or more instances ofthe compiled second workflow.

In some embodiments, the non-transitory computer readable storage mediumfurther comprises instructions for, at each respective time step in arecurring series of time steps, simulating a remainder of each of theone or more instances of the compiled first workflow thereby forming oneor more first simulations. The non-transitory computer readable storagemedium further comprises instructions for, at each respective time stepin a recurring series of time steps, simulating a remainder of each ofthe one or more instances of the compiled second workflow therebyforming one or more second simulations. In such embodiments, thenon-transitory computer readable storage medium further comprisesinstructions for firing an interlocking condition error handlerassociated with a first unit operation in an instance of the one or moreinstances of the compiled first workflow that forms an interlockingcondition with a second unit operation in an instance of the one or moreinstances of the compiled second workflow.

In some embodiments, firing the interlocking condition error handleradjusts one or more time intervals of one or more unit operations in aninstance of the compiled first workflow or an instance of the compiledsecond workflow that have not been executed.

In some embodiments, firing the interlocking condition error handleradjusts a weight one or more unit operations in an instance of thecompiled first workflow or an instance of the compiled second workflowthat have not been executed as a function of a priority assigned to thecompiled first workflow versus a priority assigned to the compiledsecond workflow.

In some embodiments, firing the interlocking condition error handleradjusts one or more time intervals of one or more unit operations in aninstance of the compiled first workflow or an instance of the compiledsecond workflow that have not been executed as a function of a priorityassigned to the compiled first workflow versus a priority assigned tothe compiled second workflow.

In some embodiments, firing the interlocking condition error handleraborts an instance of the compiled first workflow or an instance of thecompiled second workflow.

In some embodiments, the interlocking condition error handler is amutual exclusion error handler.

In some embodiments, the interlocking condition error handler suspendsan instance of the compiled first workflow or an instance of thecompiled second workflow.

In some embodiments, each time step in the recurring series of timesteps occurs on a periodic basis.

In some embodiments, each time step in the recurring series of timesteps occurs responsive to an occurrence of event in a plurality ofevent classes. In some embodiments, the event class is an instrumenterror, a power failure, a sample dropping, or an interlocking condition.

In some embodiments, each time step in the recurring series of timesteps occurs every five minutes. In some embodiments, each time step inthe recurring series of time steps occurs every 30 seconds, everyminute, every 15 minutes, every 30 minutes, or every hour.

In some embodiments, the non-transitory computer readable storage mediumfurther comprises instructions for concurrently executing two or moreinstances of the compiled first workflow and two or more instances ofthe compiled second workflow. In some embodiments, the non-transitorycomputer readable storage medium further comprises instructions forconcurrently executing three or more instances of the compiled firstworkflow and three or more instances of the compiled second workflow.

In some embodiments, the non-transitory computer readable storage mediumfurther comprises instructions, for each integer kin the set {1, k, . .. , n}, wherein n is a positive integer of two or greater, to obtain ak^(th) plurality of organic engineering targets and to assign the k^(th)plurality of organic engineering targets to a k^(th) uncompiledworkflow. The k^(th) uncompiled workflow is configured to produce thek^(th) plurality organic engineering targets, and the k^(th) uncompiledworkflow is associated with a k^(th) subset of process modules in theplurality of process modules. The instructions further cause the firstdevice to translate, for each respective organic engineering target inthe k^(th) plurality of organic engineering targets, the k^(th)uncompiled workflow into a corresponding instance of a compiled k^(th)workflow for the respective organic engineering target. Thecorresponding instance of the compiled k^(th) workflow comprises, foreach respective instrument in the k^(th) subset of instruments, anaddress of the respective instrument and one or more executioninstructions for the respective instrument as well as a k^(th) pluralityof unit operations. The k^(th) plurality of unit operations istemporally organized into a k^(th) linear temporal order, and eachrespective unit operation in the k^(th) plurality of unit operations ischaracterized by the time interval of the corresponding unit operationdefinition. A time interval of a unit operation in the k^(th) pluralityof unit operations is adjusted from the corresponding unit operationdefinition by an amount in accordance with a determination of aninterlocking condition with a unit operation in the first compiledworkflow and a unit operation in a second compiled workflow, therebyforming a plurality of instances of the compiled k^(th) workflow.

In some embodiments, the first subset of instruments comprises two ormore different instrument classes, and the second subset of instrumentscomprises two or more different instrument classes.

In some embodiments, a first instrument class and a second instrumentclass is used by both the plurality of instances of the compiled firstworkflow and the plurality of instances of the compiled second workflow.The first instrument class has a first multiplex value, and the secondinstrument class has a second multiplex value, other than the firstmultiplex value. Furthermore, the non-transitory computer readablestorage medium stores instructions for enacting a scheduler thatmaximizes a number of instances of the plurality of instances of thecompiled first workflow, a number of instances of the plurality ofinstances of the compiled second workflow, or a number of instances of acombination of instances of the compiled first workflow and the compiledsecond workflow that can concurrently use instruments of the firstinstrument class and instruments of the second instrument class giventhe first multiplex value and the second multiplex value.

In some embodiments, the scheduler maximizes, at least in part, byinvoking a first number of instances of the first instrument class as afunction of the first multiplex value of the first instrument class andinvoking a second number of instances of the second instrument class asa function of the second multiplex value of the second instrument classto be run concurrently support concurrently running instances of thecompiled first workflow and the compiled second workflow.

In some embodiments, the scheduler maximizes, at least in part, byconcurrently running a first number of instances of the first compiledworkflow and a second number of instances of the second compiledworkflow.

In some embodiments, the scheduler maximizes, at least in part, byadjusting, by an amount, a time interval of a respective unit operationin the first plurality of unit operations of an instance of the firstcompiled workflow from the time interval of the corresponding unitoperation definition or by adjusting, by an amount, a time interval of arespective unit operation in the second plurality of unit operations ofan instance of the second compiled workflow from the time interval ofthe corresponding unit operation definition.

In some embodiments, the method further comprises instructions toconcurrently execute two or more of the plurality of instances of thecompiled first workflow and two or more of the plurality of instances ofthe compiled second workflow.

In some embodiments, the method further comprises instructions toconcurrently execute two or more of the plurality of instances of thecompiled first workflow and two or more of the plurality of instances ofthe compiled second workflow. In such embodiments, the first subset ofinstruments comprises two or more instruments, the second subset ofinstruments comprises two or more instruments, and at least oneinstrument in the first subset of instruments is in the second subset ofinstruments.

In some embodiments, the method further comprises instructions toconcurrently execute three or more of the plurality of instances of thecompiled first workflow and three or more of the plurality of instancesof the compiled second workflow. In such embodiments, the first subsetof instruments comprises three or more instruments, the second subset ofinstruments comprises three or more instruments, and at least twoinstruments in the first subset of instruments is in the second subsetof instruments.

In some embodiments, two or more instances of the compiled firstworkflow are being executed at a time when the translating is executed.

In some embodiments, the non-transitory computer readable storage mediumfurther stores instructions for converting a first organic engineeringtarget in the first plurality of organic engineering targets into one ormore first inputs for the first uncompiled workflow.

In some embodiments, the first organic engineering target is synthesisof a first nucleic acid and the one or more first inputs for the firstuncompiled workflow are a set of nucleic acid bases for synthesizing thefirst nucleic acid.

In some embodiments, the first uncompiled workflow includes a branchcondition, a loop condition or a nested condition, and wherein thetranslating resolves a value associated with the branch condition, theloop condition or the nested condition in order to form the lineartemporal order of the first plurality of unit operations.

Another aspect of the present disclosure provides methods ofimplementing workflows at a first device comprising one or moreprocessors, memory storing one or more programs for execution by the oneor more processors, a controller, a communications interface, a powersupply, and one or more peripheral devices. The one or more programssingularly or collectively use the one or more processors to execute amethod. The method comprises obtaining, via the one or more peripheraldevices, a first plurality of organic engineering targets and assigning,via the controller, the first plurality of organic engineering targetsto a first uncompiled workflow. The first uncompiled workflow isconfigured to produce the first plurality of organic engineeringtargets. The first uncompiled workflow is associated with a first subsetof process modules in a plurality of process modules. Each respectiveprocess module in the plurality of process modules is associated with adifferent subset of unit operation definitions in a plurality of unitoperation definitions. Each respective unit operation definition in theplurality of unit operation definitions is independently associated witha corresponding time interval. Each respective unit operation definitionin the plurality of unit operations is independently associated with afirst subset of instruments in a plurality of instruments. The methodsfurther include translating, via the controller, for each respectiveorganic engineering target in the first plurality of organic engineeringtargets, the first uncompiled workflow into a corresponding instance ofa compiled first workflow for the respective organic engineering target.The corresponding instance of the compiled first workflow comprises, foreach respective instrument in the first subset of instruments, anaddress of the respective instrument and one or more executioninstructions for the respective instrument, as well as a first pluralityof unit operations. The first plurality of unit operations is temporallyorganized into a linear temporal order. Each respective unit operationin the first plurality of unit operations is characterized by the timeinterval of the corresponding unit operation definition. In this way, aplurality of instances of the compiled first workflow are formed. Themethods further comprise obtaining, via the one or more peripheraldevices, a second plurality of organic engineering targets andassigning, via the controller, the second plurality of organicengineering targets to a second uncompiled workflow. The seconduncompiled workflow is configured to produce the second pluralityorganic engineering targets. The second uncompiled workflow isassociated with a second subset of process modules in the plurality ofprocess modules. Furthermore, the method includes translating, via thecontroller, for each respective organic engineering target in the secondplurality of organic engineering targets, the second uncompiled workflowinto a corresponding instance of a compiled second workflow for therespective organic engineering target. The corresponding instance of thecompiled second workflow comprises, for each respective instrument inthe second subset of instruments, an address of the respectiveinstrument and one or more execution instructions for the respectiveinstrument, and a second plurality of unit operations. The secondplurality of unit operations is temporally organized into a lineartemporal order. Each respective unit operation in the second pluralityof unit operations is characterized by the time interval of thecorresponding unit operation definition. Furthermore, the methodincludes adjusting, via the controller, a time interval of a unitoperation in the second plurality of unit operations from a timeinterval of the corresponding unit operation definition by an amount inaccordance with a determination of an interlocking condition with a unitoperation in the first compiled workflow. In this way, a plurality ofinstances of the compiled second workflow are formed.

Another aspect of the present disclosure provides systems forimplementing workflows comprising a first device. The first devicecomprises a display, a power supply, a communications interface, one ormore peripheral devices, one or more processors, memory, and one or moreprograms non-transiently stored in the memory. The one or more programsare configured to be executed by the one or more processors. The one ormore programs include instructions for obtaining a first plurality oforganic engineering targets and assigning the first plurality of organicengineering targets to a first uncompiled workflow. The first uncompiledworkflow is configured to produce the first plurality of organicengineering targets. The first uncompiled workflow is associated with afirst subset of process modules in a plurality of process modules. Eachrespective process module in the plurality of process modules isassociated with a different subset of unit operation definitions in aplurality of unit operation definitions. Each respective unit operationdefinition in the plurality of unit operation definitions isindependently associated with a corresponding time interval. Eachrespective unit operation definition in the plurality of unit operationsis independently associated with a first subset of instruments in aplurality of instruments. The one or more programs further includeinstructions for translating, for each respective organic engineeringtarget in the first plurality of organic engineering targets, the firstuncompiled workflow into a corresponding instance of a compiled firstworkflow for the respective organic engineering target. Thecorresponding instance of the compiled first workflow comprises, foreach respective instrument in the first subset of instruments, anaddress of the respective instrument and one or more executioninstructions for the respective instrument, as well as a first pluralityof unit operations. The first plurality of unit operations is temporallyorganized into a linear temporal order, and each respective unitoperation in the first plurality of unit operations is characterized bythe time interval of the corresponding unit operation definition. Inthis way, a plurality of instances of the compiled first workflow areformed. The one or more programs further include instructions forobtaining a second plurality of organic engineering targets andassigning the second plurality of organic engineering targets to asecond uncompiled workflow. The second uncompiled workflow is configuredto produce the second plurality organic engineering targets. The seconduncompiled workflow is associated with a second subset of processmodules in the plurality of process modules. The one or more programsfurther include instructions for translating, for each respectiveorganic engineering target in the second plurality of organicengineering targets, the second uncompiled workflow into a correspondinginstance of a compiled second workflow for the respective organicengineering target. The corresponding instance of the compiled secondworkflow comprises, for each respective instrument in the second subsetof instruments, an address of the respective instrument and one or moreexecution instructions for the respective instrument, as well as asecond plurality of unit operations. The second plurality of unitoperations is temporally organized into a linear temporal order and eachrespective unit operation in the second plurality of unit operations ischaracterized by the time interval of the corresponding unit operationdefinition. Furthermore, the one or more programs further includeinstructions for adjusting a time interval of a unit operation in thesecond plurality of unit operations from a time interval of thecorresponding unit operation definition by an amount in accordance witha determination of an interlocking condition with a unit operation inthe first compiled workflow, thereby forming a plurality of instances ofthe compiled second workflow.

Another aspect of the present disclosure provides methods forimplementing workflows comprising a first device. The first devicecomprises a display, a power supply, a communications interface, one ormore peripheral devices, one or more processors, memory, and one or moreprograms non-transiently stored in the memory. The one or more programsare configured to be executed by the one or more processors. The one ormore programs include instructions for obtaining a first plurality oforganic engineering targets and assigning the first plurality of organicengineering targets to a first uncompiled workflow. The first uncompiledworkflow is configured to produce the first plurality of organicengineering targets. The first uncompiled workflow is associated with afirst subset of process modules in a plurality of process modules. Eachrespective process module in the plurality of process modules isassociated with a different subset of unit operation definitions in aplurality of unit operation definitions. Each respective unit operationdefinition in the plurality of unit operation definitions isindependently associated with a corresponding time interval. Eachrespective unit operation definition in the plurality of unit operationsis independently associated with a first subset of instruments in aplurality of instruments. The one or more programs further includeinstructions for translating, for each respective organic engineeringtarget in the first plurality of organic engineering targets, the firstuncompiled workflow into a corresponding instance of a compiled firstworkflow for the respective organic engineering target. Thecorresponding instance of the compiled first workflow comprises, foreach respective instrument in the first subset of instruments, anaddress of the respective instrument, and one or more executioninstructions for the respective instrument, as well as a first pluralityof unit operations. The address of the respective instrument includes acoarse grain address that is associated with a marker of the respectiveinstrument. One or more instructions are executed, which includes acoarse traverse instruction and a fine traverse instruction. The coarsetraverse instruction commands the transporter to traverse to therespective marker of the respective instrument. The fine traverseinstruction commands an articulated handling robot associated with thetransporter to move to at least one spatial coordinate associated withthe respective instrument (e.g., to move the articulated handlingrobot). The first plurality of unit operations is temporally organizedinto a linear temporal order, and each respective unit operation in thefirst plurality of unit operations is characterized by the time intervalof the corresponding unit operation definition. In this way, a pluralityof instances of the compiled first workflow are formed.

In some embodiments, the transporter includes a positioning system thatis configured to locate and guide the transporter within the transportpath.

In some embodiments, the positioning system includes a globalpositioning system. Accordingly, the respective marker of the respectiveinstrument is a global positioning system coordinate.

In some embodiments, the positioning system includes a representation(e.g., a map) of a surrounding environment of the transport path.Moreover, in some embodiments the representation of the markerpositioning of the transport path is either an image feed or a videofeed, which can have either a low-pass filter or a Gaussian blurringfunction is applied to the representation of the marker positioning ofthe transport path.

In some embodiments, the transporter includes an inertial measurementunit that is configured to verify and correct each execution of thecoarse traverse instruction of the respective instruments.

In some embodiments, the articulated handling robot includes a varietyof sensors. Each sensor provides information related to a spatiallocation of the articulated handling robot and a surrounding environmentof the articulated handling robot.

In some embodiments, the transporter includes aproportional-integral-derivative (PID) controller. The PID controllercommunicates with the sensors in order to maintain a spatial location ofthe transporter through a control loop.

In some embodiments, the articulated handling robot transfers a firsttray to a first instrument using a safety transfer procedure. The safetytransfer procedure is activated, or enabled, when a readout from thesensors fails to satisfy a threshold proximity value with respect to anactual location of the first instrument. The articulated handling robotactivates, or enables, a default transfer procedure when a readout fromthe sensors satisfies a threshold proximity value with respect to theactual physical location of the first instrument. In some embodiments,the threshold proximity value is a distance (e.g., millimeters) or adegree of rotation/orientation (e.g., degrees or Grad), or a combinationthereof.

Yet another aspect of the present disclosure provides systems forimplementing workflows, which includes a first device. The first deviceincludes a display, a power supply, a communications interface, and oneor more peripheral devices, which includes a reagent handling device.The reagent handling device includes a wash manifold, a primary valvethat regulates an internal flow path in the reagent handling device, andvariety of reagent containers. Each reagent container includes a valvethat controls a flow from the respective reagent container to theprimary valve. Moreover, the reagent handling device includes asterilization container that holds a sterilizing fluid. Similar to thereagent containers, the sterilization container includes a valve thatregulates a flow of the sterilization fluid either to the primary valveor to the wash manifold. The wash manifold is coupled to the respectivevalve of each reagent container, and regulates the flow of thesterilization fluid from the sterilization container to the reagentcontainers. Furthermore, a dispense manifold is coupled to the primaryvalve. The dispense manifold includes a variety of dispensers, andcontrol a flow from the primary valve to the dispensers. The firstdevice also includes one or more processors, memory, and one or moreprograms. The one or more programs are stored in the memory and areconfigured to be executed by the one or more processors. The one or moreprograms are configured to be executed by the one or more processors.The one or more programs include instructions for obtaining a firstplurality of organic engineering targets and assigning the firstplurality of organic engineering targets to a first uncompiled workflow.The first uncompiled workflow is configured to produce the firstplurality of organic engineering targets. The first uncompiled workflowis associated with a first subset of process modules in a plurality ofprocess modules. Each respective process module in the plurality ofprocess modules is associated with a different subset of unit operationdefinitions in a plurality of unit operation definitions. Eachrespective unit operation definition in the plurality of unit operationdefinitions is independently associated with a corresponding timeinterval. Each respective unit operation definition in the plurality ofunit operations is independently associated with a first subset ofinstruments in a plurality of instruments. The one or more programsfurther include instructions for translating, for each respectiveorganic engineering target in the first plurality of organic engineeringtargets, the first uncompiled workflow into a corresponding instance ofa compiled first workflow for the respective organic engineering target.The corresponding instance of the compiled first workflow comprises, foreach respective instrument in the first subset of instruments, anaddress of the respective instrument, and one or more executioninstructions for the respective instrument, as well as a first pluralityof unit operations.

In some embodiments, a pressure regulator is coupled to each of thesterilization container, the primary valve, and each reagent container.Furthermore, in some embodiments each pressure regulator furtherincludes a filter that prevents contamination. Moreover, in someembodiments, each pressure regulator further includes a check valve thatprevents evaporation or vapor release of a reagent.

In some embodiments, the reagent handling device includes a pump that iscoupled to the primary valve. The pump drives the internal flow path ofthe reagent handling device.

In some embodiments, the reagent handling device includes a dispensingcycle. The dispensing cycle dispenses a selection of reagents from a setof reagent containers. In some embodiments, the reagent handling deviceincludes a first sterilization cycle, which sterilizes a portion of thesystem. Similarly, in some embodiments the reagent handling deviceincludes a second sterilization cycle that purges a portion of thesystem.

In some embodiments, each component (e.g., each container, valve, etc.)of the reagent handling device is coupled through removable tubing.

In some embodiments, human intervention is only required to replenish areagent container and/or to replenish the sterilization fluid of thesterilization container.

Yet another aspect of the present disclosure provides methods forimplementing workflows comprising a first device. The first devicecomprises a display, a power supply, a communications interface, one ormore peripheral devices, one or more processors, memory, and one or moreprograms non-transiently stored in the memory. The one or more programsare configured to be executed by the one or more processors. The one ormore programs include instructions for obtaining a first plurality oforganic engineering targets and assigning the first plurality of organicengineering targets to a first uncompiled workflow. The first uncompiledworkflow is configured to produce the first plurality of organicengineering targets. The first uncompiled workflow is associated with afirst subset of process modules in a plurality of process modules. Eachrespective process module in the plurality of process modules isassociated with a different subset of unit operation definitions in aplurality of unit operation definitions. Each respective unit operationdefinition in the plurality of unit operation definitions isindependently associated with a corresponding time interval. Eachrespective unit operation definition in the plurality of unit operationsis independently associated with a first subset of instruments in aplurality of instruments. The one or more programs further includeinstructions for translating, for each respective organic engineeringtarget in the first plurality of organic engineering targets, the firstuncompiled workflow into a corresponding instance of a compiled firstworkflow for the respective organic engineering target. Thecorresponding instance of the compiled first workflow comprises, foreach respective instrument in the first subset of instruments, anaddress of the respective instrument, and one or more executioninstructions for the respective instrument, as well as a first pluralityof unit operations. The one or more execution instructions includeinstructions for a multi-well plate centrifuge instrument. Theseinstructions include determining a mass of each multi-well plate in afirst set of multi-well plates. Each multi-well plate is disposed intothe multi-well plate centrifuge. Furthermore, each respective multi-wellplate has a corresponding counter balance in a set of counter balances,which is disposed in the multi-well plate centrifuge at a positionopposite the respective multi-well plate. Without human intervention, amass of each respective counter balance is adjusted to be equal to thecorresponding multi-well plate. The multi-well centrifuge is thenoperated. The first plurality of unit operations is temporally organizedinto a linear temporal order, and each respective unit operation in thefirst plurality of unit operations is characterized by the time intervalof the corresponding unit operation definition. In this way, a pluralityof instances of the compiled first workflow are formed.

In some embodiments, the first set of multi-well plates is a singlemulti-well plate.

In some embodiments, the first set of multi-well plates is a set of fromtwo multi-well plates to five multi-well plates.

In some embodiments, the adjusting includes pumping fluid (e.g., water,mineral oil, gas) to the respective counter balance in order to adjustthe mass therein.

In some embodiments, the adjusting includes drawing fluid from therespective counter balance in order to adjust the mass therein.

In some embodiments, each counter balance includes a bottom end portionthat is configured to pool fluid therein.

In some embodiments, the determining and the adjusting are performedsimultaneously.

In some embodiments, the adjusting includes storing the adjusted mass ofeach counter balance.

In some embodiments, the one or more instructions for the multi-wellplate centrifuge instrument includes determining a mass of eachmulti-well plate in a second set of multi-well plates, disposing eachmulti-well plate in the second set into the centrifuge, adjusting,without human intervention, the mass of each counter balance in a secondset of counter balances to be equal to the mass of the correspondingmulti-well plate in the second set of multi-well plates, and operatingthe multi-well plate centrifuge.

In some embodiments, the second set of multi-well plates are the firstset of multi-well plates.

The automated biological foundry of the present invention has otherfeatures and advantages that will be apparent from, or are set forth inmore detail in, the accompanying drawings, which are incorporatedherein, and the following Detailed Description, which together serve toexplain certain principles of exemplary embodiments of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, and 1D illustrate a computer system in accordance withan embodiment of the present disclosure;

FIGS. 2A, and 2B illustrate a system topology and hardware layout inaccordance with various embodiments of the present disclosure;

FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, and 3L collectivelyillustrate a flow chart of methods for supporting automated workflowsusing a first device in accordance with an embodiment of the presentdisclosure, in which optional steps or embodiments are indicated bydashed boxes;

FIG. 4A illustrates an overall design for single-transcript TALENsynthesis according to an embodiment of the present disclosure;

FIG. 4B illustrates an assembly scheme of the design and preliminarytest of single-transcript TALEN synthesis according to an embodiment ofthe present disclosure;

FIG. 4C illustrates a test assembly of a single-transcript TALEN pairaccording to an embodiment of the present disclosure;

FIG. 5A illustrates a single-transcript expression of a TALEN pairaccording to an embodiment of the present disclosure;

FIG. 5B illustrates genome editing in HEK293T cells according to anembodiment of the present disclosure;

FIGS. 5C, 5D, and 5E illustrate disruption of an Oct4 enhancer in H1hESC according to an embodiment of the present disclosure;

FIGS. 6A and 6B illustrate a breakdown of unit operations of an iBioFABsystem according to an embodiment of the present disclosure;

FIGS. 6C and 6D illustrate various control hierarchies of an iBioFABsystem according to embodiments of the present disclosure;

FIG. 7A illustrates general workflow for the DNA assembly pipeline basedon Golden Gate method according to an embodiment of the presentdisclosure;

FIG. 7B illustrates a process flow diagram for the build step accordingto an embodiment of the present disclosure;

FIG. 7C and FIG. 7D illustrate verification of single-transcript TALENssynthesized in high throughput according to an embodiment of the presentdisclosure;

FIGS. 8A and 8B illustrate plasmid design for single plasmid TALENassembly according to an embodiment of the present disclosure;

FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, and 9H illustrate disrupting EGFP inHEK293 cells according to an embodiments of the present disclosure;

FIG. 10 illustrates a Gantt chart for automated Golden Gate DNA assemblyworkflow according to an embodiment of the present disclosure;

FIG. 11A and FIG. 11B illustrate a list of substrates according to anembodiment of the present disclosure;

FIG. 12 illustrates a list of results from T7E1 assay according to anembodiment of the present disclosure;

FIG. 13 illustrates a fluid management system according to an embodimentof the present disclosure;

FIGS. 14A, 14B, and 14C illustrate a centrifuge compatible vacuummanifold according to an embodiment of the present disclosure;

FIG. 15 illustrates a pipette tip according to an embodiment of thepresent disclosure;

FIGS. 16A and 16B illustrate a dispending pattern and streakingpatterning according to an embodiment of the present disclosure;

FIGS. 16C and 16D illustrate another dispending pattern and streakingpatterning according to an embodiment of the present disclosure;

FIGS. 17A and 17B illustrate a streaking cone according to an embodimentof the present disclosure;

FIG. 18 illustrates an automated centrifuge handling system according toan embodiment of the present disclosure; and

FIG. 19 illustrates a positioning system according to an embodiment ofthe present disclosure;

The specific design features of the present invention as disclosedherein, including, for example, specific dimensions, orientations,locations, and shapes will be determined in part by the particularintended application and use environment.

In the figures, reference numbers refer to the same or equivalent partsof the present invention throughout the several figures of the drawing.

DETAILED DESCRIPTION

Reference will now be made in detail to various embodiments of thepresent invention(s), examples of which are illustrated in theaccompanying drawings and described below. While the invention(s) willbe described in conjunction with exemplary embodiments, it will beunderstood that the present description is not intended to limit theinvention(s) to those exemplary embodiments. On the contrary, theinvention(s) is/are intended to cover not only the exemplaryembodiments, but also various alternatives, modifications, equivalentsand other embodiments, which may be included within the spirit and scopeof the invention as defined by the appended claims.

As used herein, in some embodiments, the term “set” means two or more,three or more, or four or more.

It will also be understood that, although the terms first, second, etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. For example, a first workflow could be termeda second workflow, and, similarly, a second workflow could be termed afirst workflow, without departing from the scope of the presentdisclosure. The first workflow and the second workflow are bothworkflows, but they are not the same workflow.

The terminology used in the present disclosure is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. As used in the description of the inventionand the appended claims, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will also be understood that the term “and/or”as used herein refers to and encompasses any and all possiblecombinations of one or more of the associated listed items. It will befurther understood that the terms “comprises” and/or “comprising,” whenused in this specification, specify the presence of stated features,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,integers, steps, operations, elements, components, and/or groupsthereof.

In the interest of clarity, not all of the routine features of theimplementations described herein are shown and described. It will beappreciated that, in the development of any such actual implementation,numerous implementation-specific decisions are made in order to achievethe designer's specific goals, such as compliance with use case- andbusiness-related constraints, and that these specific goals will varyfrom one implementation to another and from one designer to another.Moreover, it will be appreciated that such a design effort might becomplex and time-consuming, but nevertheless be a routine undertaking ofengineering for those of ordering skill in the art having the benefit ofthe present disclosure.

As used herein, the term “if” may be construed to mean “when” or “upon”or “in response to determining” or “in response to detecting,” dependingon the context. Similarly, the phrase “if it is determined” or “if [astated condition or event] is detected” may be construed to mean “upondetermining” or “in response to determining” or “upon detecting [thestated condition or event]” or “in response to detecting [the statedcondition or event],” depending on the context.

In some embodiments, systems and methods for supporting automatedworkflows in accordance with the present disclosure obtain a first setof targets and assign these targets to a first uncompiled workflow type.This first uncompiled workflow type is configured to produce thetargets. Moreover, the first uncompiled workflow comprise processmodules each of which is further associated with a subset of unitoperation definitions. Each unit operation definition is associated witha time interval. Each unit operation is further associated with a subsetof instruments. The present disclosure translates the first uncompiledworkflow, for each target in the first set of targets, into an instanceof a first compiled workflow. The instance of the first compiledworkflow comprises an address of the instruments and executioninstructions for the instruments. The unit operations are organized intoa linear temporal order.

The systems and methods of the present disclosure further supportobtaining a set of second targets and assigning them to an uncompiledsecond workflow. This uncompiled second workflow may be the same ordifferent then the uncompiled first workflow. The uncompiled secondworkflow is configured to produce the second targets. The uncompiledsecond workflow is associated with different process modules from thefirst uncompiled workflow. The second uncompiled workflow is translatedinto an instance of a compiled second workflow for each respectivetarget in the set of second targets. Moreover, a time interval of unitoperations in the second workflow is adjusted from the correspondingunit operation definition by an amount in determination of aninterlocking condition with a unit operation in the first compiledworkflow. In this way, one or more of the second compiled workflows canbe executed on the same foundry at the same time as one or more of thefirst compiled workflows are executed. In fact, in some embodiments, twoor more of the second compiled workflows are executed on the samefoundry at the same time as two or more of the first compiled workflows.

In this way, multiple workflows can be run on the same foundry in anefficient manner. Although mechanisms for compiling two different typesof workflows and running them on the same foundry have been disclosed,the present disclosure is not so limited. In some embodiments, two ormore instances of three or more, four or more, five or more, ten ormore, twenty or more, or one hundred or more different types of compiledworkflows are concurrently run on the same foundry by adjusting the timeinterval of unit operations in the respective workflows to avoidinterlocking conditions.

FIG. 1 details just such an exemplary system 11 for use in supportingmultiple workflows in a biological foundry. The system preferablycomprises a computer system 10 having:

-   -   a central processing unit (CPU) 22;    -   a main non-volatile (non-transitory) storage unit 14, for        example a hard disk drive, for storing software and data, the        storage unit 14 controlled by storage controller 12;    -   a system memory 36, preferably high speed random-access memory        (RAM), for storing system control programs, data, and        application programs, comprising programs and data loaded from        non-volatile storage unit 14; system memory 36 may also include        read-only memory (ROM);    -   a user interface 32, comprising one or more input devices (e.g.,        keyboard 28, a mouse) and a display 26 or other output device;    -   optionally, a network interface card 20 (communications        interface) for connecting to any wired or wireless communication        network 34 (e.g., a wide area network such as the Internet);    -   a power source 24 to power the aforementioned elements; and    -   an internal bus 30 for interconnecting the aforementioned        elements of the system.

Operation of computer 10 is controlled primarily by operating system 40,which is executed by central processing unit 22. Operating system 40 canbe stored in system memory 36. In a typical implementation, systemmemory 36 also includes:

-   -   a file system 42 for controlling access to the various files and        data structures;    -   unit operation definitions 44 which includes execution        instructions for a plurality of instruments and physical or        chemical procedures to impart on the organic engineering targets        conducted by a single instrument;    -   instruments 46 including addresses of each instrument;    -   laboratory information management system 48 which includes        features support modules to manage operations of a laboratory;    -   an engineering target library 50 comprising tables of plausible        and/or stored engineering targets;    -   a workflow library 52 comprising a table of predetermined        workflows, workflow templates, and stored workflows;    -   a process module library 54 comprising a table of predetermined        process modules and stored process modules;    -   a scheduler 56 which assists in managing and organizing        operations of workflows; and    -   compiled workflows 58 comprising the data of compiled workflows.

As illustrated in FIG. 1, computer 10 comprises data such as unitoperation definitions 44, engineering target library 50, workflowlibrary 52 and the like. Such data can be stored in any form of datastorage system including, but not limited to, a flat file, a relationaldatabase (SQL), or an on-line analytical processing (OLAP) database (MDXand/or variants thereof). In some embodiments, as associated data isstored in a single database. In other embodiments, as well as associateddata is stored in a plurality of databases that may or may not all behosted by the same computer 10. In such embodiments, some components aswell as associated data are stored on computer systems that are notillustrated by FIG. 1 but that are addressable by wide area network 34.

In some embodiments, unit operation definitions 44 as well as associateddata for such instruments 46, engineered target library 50, workflowlibrary 52, process modules 54, and related software modules illustratedin FIG. 1 are on a single computer (e.g., computer 10) and in otherembodiments they are hosted by several computers (not shown). In fact,all possible arrangements of unit operation definitions 44, instruments46, engineered target library 50, workflow library 52, process modules54, and the modules illustrated in FIG. 1 on one or more computers arewithin the scope of the present disclosure so long as these componentsare addressable with respect to each other across computer network 34 orby other electronic means. Thus, the present disclosure fullyencompasses a broad array of computer systems.

Now that a system has been described for supporting multiple automatedworkflows in accordance with various exemplary embodiments of thepresent disclosure, details regarding some processes in accordance withFIG. 3 will be disclosed. FIG. 3 collectively illustrates a flow chartof methods for supporting multiple automated workflows in accordancewith an exemplary embodiment of the present disclosure. In the flowchart, the preferred parts of the methods are shown in solid line boxeswhereas optional variants of the methods, or optional equipment used bythe methods, are shown in dashed line boxes. As such, FIG. 3 illustratesmethods for supporting multiple automated workflows.

Certain steps are performed by various modules in memory 36. It will beappreciated that the steps described in FIG. 3 can be encoded in asingle module or any combination of modules.

In describing the methods of FIG. 3, a first workflow and a secondworkflow are described in many embodiments. It should be appreciated,however, that in accordance with the present disclosure there can, foreach integer kin the set {1, . . . , k, . . . , n}, where n is apositive integer of two or greater, exist n total workflows.Additionally, n refers to a maximum number in a given set. Thus a k^(th)workflow is a generic workflow in the set of n workflows. As such, atheoretical limit, or bottleneck, to a number of active workflows is anumber of instruments in a given system or a throughput of a giveninstrument or class of instruments.

Referring to blocks 1002-1006 of FIG. 3A, a method for implementingworkflows will now be described. At a first device (e.g., computer 10 ofFIG. 1) comprising one or more processors, memory storing one or moreprograms for execution by the one or more processors, a controller(e.g., controller 12 of FIG. 1), a communications interface (e.g.,communications circuity 20 of FIG. 1), a power supply (e.g., powersource 24 of FIG. 1), and one or more peripheral devices (e.g., keyboard28 and display 26 of FIG. 1), the one or more programs singularly orcollectively executing a given method.

In some embodiments, the first device communicates with at least oneexternal control server or external database server. In someembodiments, data is saved by the first device which describes data ofthe workflow and/or executed instructions. For instance, in someembodiments the data is exported as one or more tab delimited files, CSVfiles, EXCEL spreadsheets, GOOGLE Sheets, or in a form suitable for anSQL database. Additionally, such communication can be utilized for aplurality of purposes, including, but not limited to, communicating withfirst devices of other systems, saving data of a workflow to an externalwebserver or database server, saving data which describes the executedinstructions of instruments, or the like. Examples of networks include,but are not limited to, the World Wide Web (WWW), an intranet and/or awireless network, such as a cellular telephone network, a wireless localarea network (LAN) and/or a metropolitan area network (MAN), and otherdevices by wireless communication. The wireless communication optionallyuses any of a plurality of communications standards, protocols andtechnologies, including but not limited to Global System for MobileCommunications (GSM), Enhanced Data GSM Environment (EDGE), high-speeddownlink packet access (HSDPA), high-speed uplink packet access (HSUPA),Evolution, Data-Only (EV-DO), HSPA, HSPA+, Dual-Cell HSPA (DC-HSPDA),long term evolution (LTE), near field communication (NFC), wideband codedivision multiple access (W-CDMA), code division multiple access (CDMA),time division multiple access (TDMA), Bluetooth, Wireless Fidelity(Wi-Fi) (e.g., IEEE 802.11a, IEEE 802.11ac, IEEE 802.11ax, IEEE 802.11b,IEEE 802.11g and/or IEEE 802.11n), voice over Internet Protocol (VoIP),Wi-MAX, a protocol for e-mail (e.g., Internet message access protocol(IMAP) and/or post office protocol (POP)), instant messaging (e.g.,extensible messaging and presence protocol (XMPP), Session InitiationProtocol for Instant Messaging and Presence Leveraging Extensions(SIMPLE), Instant Messaging and Presence Service (IMPS)), and/or ShortMessage Service (SMS), or any other suitable communication protocol,including communication protocols not yet developed as of the filingdate of this document (1004, 1006).

Referring to blocks 1008 through 1012 of FIG. 3A, the method furtherrequires obtaining, via the one or more peripheral devices (e.g.,keyboard 28 of FIG. 1), a first plurality of organic engineeringtargets. In some embodiments, such as automating workflows in abiological foundry, each organic engineering target in the firstplurality of organic engineering targets is a plurality of reagents ofnucleic acid components. In some embodiments, each organic engineeringtarget in the first plurality of organic engineering targets is anassembly of nucleic acid components. For instance, in some embodiments,each organic engineering target in the first plurality of organicengineering targets is a plasmid, and the nucleic acid components arepredetermined promoters, repressors, stop codon, and exons. In someembodiments, each organic engineering target in the first plurality oforganic engineering targets is a different predetermined nucleic acidwith a different predetermined nucleic acid sequence. In someembodiments, each organic engineering target in the first plurality oforganic engineering targets is a different predetermined ribonucleicacid (mRNA) with a different predetermined nucleic acid sequence. Insome embodiments, each organic engineering target in the first pluralityof organic engineering targets is a different predetermineddeoxyribonucleic acid (DNA) with a different predetermined nucleic acidsequence. In some embodiments, each organic engineering target in thefirst plurality of organic engineering targets is a differentpredetermined polymer. In some embodiments, each organic engineeringtarget in the first plurality of organic engineering targets is adifferent predetermined peptide. In some embodiments, each organicengineering target in the first plurality of organic engineering targetsis a different predetermined protein.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises a differentheteropolymer (copolymer). A copolymer is a polymer derived from two (ormore) monomeric species, as opposed to a homopolymer where only onemonomer is used. Copolymerization refers to methods used to chemicallysynthesize a copolymer. Examples of copolymers include, but are notlimited to, ABS plastic, SBR, nitrile rubber, styrene-acrylonitrile,styrene-isoprene-styrene (SIS) and ethylene-vinyl acetate. Since acopolymer consists of at least two types of constituent units (alsostructural units, or particles), copolymers can be classified based onhow these units are arranged along the chain. These include alternatingcopolymers with regular alternating A and B units. See, for example,Jenkins, 1996, “Glossary of Basic Terms in Polymer Science,” Pure Appl.Chem. 68 (12): 2287-2311, which is hereby incorporated herein byreference in its entirety. Additional examples of copolymers areperiodic copolymers with A and B units arranged in a repeating sequence(e.g. (A-B-A-B-B-A-A-A-A-B-B-B)_(n)). Additional examples of copolymersare statistical copolymers in which the sequence of monomer residues inthe copolymer follows a statistical rule. If the probability of findinga given type monomer residue at a particular point in the chain is equalto the mole fraction of that monomer residue in the chain, then thepolymer may be referred to as a truly random copolymer. See, forexample, Painter, 1997, Fundamentals of Polymer Science, CRC Press,1997, p 14, which is hereby incorporated by reference herein in itsentirety. Still other examples of copolymers that may be evaluated usingthe disclosed systems and methods are block copolymers comprising two ormore homopolymer subunits linked by covalent bonds. The union of thehomopolymer subunits may require an intermediate non-repeating subunit,known as a junction block. Block copolymers with two or three distinctblocks are called diblock copolymers and triblock copolymers,respectively.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises a plurality ofpolymers, where the respective polymers in the plurality of polymers donot all have the same molecular weight. In such embodiments, thepolymers in the plurality of polymers fall into a weight range with acorresponding distribution of chain lengths. In some embodiments, thepolymer is a branched polymer molecular system comprising a main chainwith one or more substituent side chains or branches. Types of branchedpolymers include, but are not limited to, star polymers, comb polymers,brush polymers, dendronized polymers, ladders, and dendrimers. See, forexample, Rubinstein et al., 2003, Polymer physics, Oxford; New York:Oxford University Press. p. 6, which is hereby incorporated by referenceherein in its entirety.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises a polypeptide. Asused herein, the term “polypeptide” means two or more amino acids orresidues linked by a peptide bond. The terms “polypeptide” and “protein”are used interchangeably herein and include oligopeptides and peptides.An “amino acid,” “residue” or “peptide” refers to any of the twentystandard structural units of proteins as known in the art, which includeimino acids, such as proline and hydroxyproline. The designation of anamino acid isomer may include D, L, R and S. The definition of aminoacid includes nonnatural amino acids. Thus, selenocysteine, pyrrolysine,lanthionine, 2-aminoisobutyric acid, gamma-aminobutyric acid,dehydroalanine, ornithine, citrulline and homocysteine are allconsidered amino acids. Other variants or analogs of the amino acids areknown in the art. Thus, a polypeptide may include syntheticpeptidomimetic structures such as peptoids. See Simon et al., 1992,Proceedings of the National Academy of Sciences USA, 89, 9367, which ishereby incorporated by reference herein in its entirety. See also Chinet al., 2003, Science 301, 964; and Chin et al., 2003, Chemistry &Biology 10, 511, each of which is incorporated by reference herein inits entirety.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises a polypeptide havingany number of posttranslational modifications. Thus, a polypeptideincludes those that are modified by acylation, alkylation, amidation,biotinylation, formylation, γ-carboxylation, glutamylation,glycosylation, glycylation, hydroxylation, iodination, isoprenylation,lipoylation, cofactor addition (for example, of a heme, flavin, metal,etc.), addition of nucleosides and their derivatives, oxidation,reduction, pegylation, phosphatidylinositol addition,phosphopantetheinylation, phosphorylation, pyroglutamate formation,racemization, addition of amino acids by tRNA (for example,arginylation), sulfation, selenoylation, ISGylation, SUMOylation,ubiquitination, chemical modifications (for example, citrullination anddeamidation), and treatment with other enzymes (for example, proteases,phosphotases and kinases). Other types of posttranslationalmodifications are known in the art and are also included.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises an organometalliccomplex. An organometallic complex is chemical compound containing bondsbetween carbon and metal. In some instances, organometallic compoundsare distinguished by the prefix “organo-” e.g. organopalladiumcompounds. Examples of such organometallic compounds include all Gilmanreagents, which contain lithium and copper. Tetracarbonyl nickel, andferrocene are examples of organometallic compounds containing transitionmetals. Other examples include organomagnesium compounds likeiodo(methyl)magnesium MeMgI, diethylmagnesium (Et₂Mg), and all Grignardreagents; organolithium compounds such as n-butyllithium (n-BuLi),organozinc compounds such as diethylzinc (Et₂Zn) andchloro(ethoxycarbonylmethyl)zinc (ClZ_(n)CH₂C(═O)OEt); and organocoppercompounds such as lithium dimethylcuprate (Li⁺[CuMe₂]⁻). In addition tothe traditional metals, lanthanides, actinides, and semimetals, elementssuch as boron, silicon, arsenic, and selenium are considered formorganometallic compounds, e.g. organoborane compounds such astriethylborane (Et₃B).

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises two different typesof polymers, such as a nucleic acid bound to a polypeptide. In someembodiments, the polymer includes two polypeptides bound to each other.In some embodiments, the polymer under study includes one or more metalions (e.g. a metalloproteinase with one or more zinc atoms) and/or isbound to one or more organic small molecules (e.g., an inhibitor). Insuch instances, the metal ions and or the organic small molecules may berepresented as one or more additional particles p_(i) in the set of {p₁,. . . , p_(K)} particles representing the native polymer.

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises a protein. The basicstructural elements of proteins are well-known in the art. Nonterminalamino acids typically have the structure —NH—C^(α)HR—CO—, where Rrepresents an amino acid side chain as is known in the art. Atoms suchas N, C^(α), Cº and O that are not in the sidechain represent backboneatoms. Atoms of the sidechain, especially the heteroatoms of thesidechain, are referred to as “terminal” atoms. Thus, terminal atomsinclude C^(β) in alanine, S^(γ) in cysteine, and N^(εl) and C^(ηl) intryptophan, for example. Such terminal atoms can be unique. C-alpha orC^(α) is the carbon atom in the center of each amino acid. The proteinbackbon e includes N, C-alpha, C and O atoms. The backbone dihedralangles of proteins are called ϕ (phi, involving the backbone atomsC′—N—C^(α)-C′), ψ (psi, involving the backbone atoms N—C^(α)-C′—N) and ω(omega, involving the backbone atoms C^(α)-C′—N—C^(α)). Thus, ϕ controlsthe C′-C′ distance, ψ controls the N—N distance and co controls theC^(α)-C^(α) distance. The planarity of the peptide bond usuallyrestricts co to be 180° (the typical trans case) or 0° (the rare ciscase). The sidechain dihedral angles tend to cluster near 180°, 60°, and−60°, which are called the trans, gauche⁺, and gauche⁻ conformations.The choice of sidechain dihedral angles is affected by the neighbouringbackbone and sidechain dihedrals. A Ramachandran map (Ramachandran,Ramakrishnan, and Sasisekharan 1963) is a representation of thestereochemically allowed protein backbone geometries as a function oftheir variable torsion angles.

There are different levels of describing the structure of a protein.Primary structure refers to the linear sequence of amino acids that makeup the polypeptide chain. The bond between two amino acids is a peptidebond. The sequence of amino acids determines the positioning of thedifferent R groups relative to each other. This positioning determinesthe way that the protein folds and the final structure of the molecule.The secondary structure of protein molecules refers to the formation ofa regular pattern of twists or kinks of the polypeptide chain. Theregularity is due to hydrogen bonds forming between the atoms of theamino acid backbone of the polypeptide chain. The two most common typesof secondary structure are called the “α-helix” and “β-pleated sheet”.Tertiary structure refers to the three dimensional globular structureformed by bending and twisting of the polypeptide chain. This processoften means that the linear sequence of amino acids is folded into acompact globular structure. The folding of the polypeptide chain isstabilized by multiple weak, noncovalent interactions. Theseinteractions include hydrogen bonds, electrostatic interactions,hydrophobic interactions, and sometimes covalent bonds. Quaternarystructure refers to the fact that some proteins contain more than onepolypeptide chain, adding an additional level of structuralorganization: the association of the polypeptide chains. Eachpolypeptide chain in the protein is called a subunit. The subunits canbe the same polypeptide chain or different ones. For example, the enzymeβ-galactosidase is a tetramer, meaning that it is composed of foursubunits, and, in this case, the subunits are identical—each polypeptidechain has the same sequence of amino acids. Hemoglobin, the oxygencarrying protein in the blood, is also a tetramer but it is composed oftwo polypeptide chains of one type (141 amino acids) and two of adifferent type (146 amino acids).

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets comprises a chemical compoundthat satisfies the Lipinski rule of five criteria. In some embodiments,the chemical compound is an organic compounds that satisfies two or morerules, three or more rules, or all four rules of the Lipinski's Rule ofFive: (i) not more than five hydrogen bond donors (e.g., OH and NHgroups), (ii) not more than ten hydrogen bond acceptors (e.g. N and O),(iii) a molecular weight under 500 Daltons, and (iv) a Log P under 5.The “Rule of Five” is so called because three of the four criteriainvolve the number five. See, Lipinski, 1997, Adv. Drug Del. Rev. 23, 3,which is hereby incorporated herein by reference in its entirety. Insome embodiments, the organic engineering target satisfies one or morecriteria in addition to Lipinski's Rule of Five. For example, in someembodiments, the test perturbation is a compound with five or feweraromatic rings, four or fewer aromatic rings, three or fewer aromaticrings, or two or fewer aromatic rings.

As such, in the context of biological engineering, an organicengineering target is one of the objectives of a research anddevelopment project that defines the desired biological trait to beachieved. The organic engineering target can be either quantitative orqualitative. For example, in one embodiment, an organic engineeringtarget(s) can be a genetic configuration for a biosynthetic pathway thatproduces more compound of interest than a current level. In anotherembodiment, the organic engineering target(s) is a genetic configurationfor a microbial host that has a tolerance to an inhibitor over X mg/L.Additionally, in some embodiments an organic engineering target is apolynucleotide or nucleic acid sequence. The terms “polynucleotide” and“nucleic acid sequence” interchangeably refer to a polymer composed ofnucleotide units as would be understood by one of skill in the art.Preferred nucleotide units include but are not limited to thosecomprising adenine (A), guanine (G), cytosine (C), thymine (T), anduracil (U). Useful modified nucleotide units include but are not limitedto those comprising 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine,2-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylamino-methyluridine, dihydrouridine,2-O-methylpseudouridine, 2-O-methylguanosine, inosine,N6-isopentyladenosine, 1-methyladenosine, 1-methylpseudouridine,1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, 3-methylcytidine,5-methylcytidine, N6-methyladenosine, 7-methylguanosine,5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,5-methoxyuridine, 5-methoxycarbonylmethyl-2-thiouridine,5-methoxycarbonylmethyluridine, 2-methylthio-N6-isopentyladenosine,uridine-5-oxyacetic acid-methylester, uridine-5-oxyacetic acid,wybutoxosine, wybutosine, pseudouridine, queuosine, 2-thiocytidine,5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine,2-O-methyl-5-methyluridine, 2-O-methyluridine, and the like.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”), as well asnucleic acid analogs. Nucleic acid analogs include those that includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor that include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like.

Furthermore, in some embodiments an organic engineering target refers toa polynucleotide sequence that can be assembled together to form an“engineered nucleic acid construct” using the methods of polynucleotideassembly described herein. A “component polynucleotide,” alternatelyreferred to as “bits” herein, refers to any isolated or isolatablemolecule of DNA. Useful examples include but are not limited to aprotein-coding sequence, reporter gene, fluorescent marker codingsequence, promoter, enhancer, terminator, intron, exon, poly-A tail,multiple cloning site, nuclear localization signal, mRNA stabilizationsignal, selectable marker, integration loci, epitope tag codingsequence, degradation signal, or any other naturally occurring orsynthetic DNA molecule. In some embodiments, the DNA segment is ofnatural origin. Alternatively, a DNA segment can be completely ofsynthetic origin, produced in vitro. Furthermore, a DNA segment cancomprise any combination of isolated naturally occurring DNA molecules,or any combination of an isolated naturally occurring DNA molecule and asynthetic DNA molecule. For example, a DNA segment may comprise aheterologous promoter operably linked to a protein coding sequence, aprotein coding sequence linked to a poly-A tail, a protein codingsequence linked in-frame with an epitope tag coding sequence, and thelike. Working examples of various organic engineering targets todescribed infra (1008, 1010, 1012).

Referring to block 1014 of FIG. 3A, following selection of the firstplurality of engineering targets, the method includes assigning thefirst plurality of organic engineering targets to a first uncompiledworkflow. In general, a workflow is a generalized laboratory processthat includes a series of unit operations to achieve an engineeringtarget. Workflows can be applied to different sample, or organicengineering target, batches with different parameter sets. The firstuncompiled workflow is configured to produce the first plurality oforganic engineering targets, and the first uncompiled workflow isassociated with a first subset of process modules in a plurality ofprocess modules. A process module is a generalized laboratory processthat consists of a series of unit operations. In most cases, a processmodule is routinely performed in workflows and shared by researchprojects. When developing workflows, process modules can be called froma library (e.g., process module library 54) and configured with anappropriate parameter set to simplify and standardize programmingpractice. Process modules can be nested to form complex workflows. Forinstance, referring to FIG. 6A, an exemplary evolutionary engineeringworkflow is associated with a subset of process modules including cellculture and sample, whereas a library screening workflow is associatedwith normalization, transformation, cell culture, and sample processmodules. Each respective process module in the plurality of processmodules is associated with a different subset of unit operationdefinitions in a plurality of unit operation definitions. Eachrespective unit operation definition in the plurality of unit operationdefinitions is independently associated with a corresponding timeinterval as well as each respective unit operation definition in theplurality of unit operations is independently associated with a firstsubset of instruments in a plurality of instruments. In the context ofbiological engineering, a unit operation is a basic step in a laboratoryprocess. Unit operations involve a physical or chemical procedure on thesamples conducted by a single instrument. In scheduling, a unitoperation or action is a largest inseparable unit that may consist of asequence of micro steps (e.g. no other procedure or delay can be cutinto these micro steps). As shown in FIG. 6A, an exemplary processmodule “normalization” includes the unit operation(s) pipetting, whereasthe process module “DNA quantification” includes unit operations“spectrophotometry” and “pipetting” (1014).

Subsets of instruments include but are not limited to systems and/ordevices that perform unit operations. For instance, referring to FIG.18, in some embodiments a subset of instruments includes an automatedcentrifuge handling system 1800. The automated centrifuge handing system1800 includes a robotic arm that operates various instruments of thesystem and handles samples.

In some embodiments, the robotic arm is a Cartesian robotic arm. In someembodiments, the robotic arm is an articulated robotic arm, includingfour axis robotic arms, five-axis robotic arms, six-axis robotic arms,and seven-axis robotic arms. For instance, in some embodiments therobotic arm is a Kuka KR 3 R540 AGILUS robotic arm or a Kuka KR 1000titan F robotic arm. In some embodiments, the robotic arm is a selectivecompliance assembly robot arm (SCARA). In some embodiments, the roboticarm is the previously described transporter. In some embodiments, therobotic arm is a conveyer belt, a linear transfer stage, a gantry, or anelevator.

The robotic arm 1802 transfers samples from a storage device 1804 to amulti-well plate, or similar device which is utilized by a centrifuge1818 such as a vial, a bottle, a screw-top tube, a snap-top tube, anopen tube, a flip-top tube, etc. Once the multi-well plate, or in someembodiments a multitude of multi-well plates, is filled, a mass of eachmulti-well plate is determined. In some embodiments, these multi-wellplates form a first set of multi-well plates (e.g., the multi-wellplates of a first operation of the centrifuge). A number of multi-wellplates in a set of multi-well plates is determined by a type and size ofthe centrifuge 1818. For instance, in some embodiments the centrifuge isconfigured to hold one multi-well plate, is configured to hold two setsof multi-well plates, three sets of multi-well plates, four sets ofmulti-well plates, five sets of multi-well plates, etc. A number ofmulti-well plates and a type of multi-well plate is dictated by a typeof centrifuged utilized in the present disclosure. Accordingly, in someembodiments the multi-well plates are 6-well plates, are 12-well plates,are 24-well plates, are 48 well-plates, are 96-well plates, are 384-wellplates, or are 1536-well plates. Similarly, in some embodiments theseplates are formed with a U-shaped bottom end portion, with a V-shapedbottom end portion, or with a flat-shaped bottom end portion. Forinstance, in some embodiments the multi-well plates are Fisherbrand96-well plates No.: N95029340FP; are Thermo Fisher Scientific Nunc U96Microwell Plates, PS; are Thermo Fisher Scientific Nunc V96 MicrowellPlates, Clear, PS; are CELLTREAT 96 Well Non-Treated Microplates,F-Bottom, Clear, Sterile; are iSci d75-712 Microplates; or are Corning3370 Plates, 96 well, PS, Flat, 100/cs, Clear. However, the presentdisclosure is not limited thereto.

Centrifuges of the present disclosure include, but are not limited to,small benchtop centrifuges, micro-centrifuges (e.g., a microfuge), highspeed centrifuges, or ultra-centrifuges. In some embodiments, thecentrifuge is an Eppendorf 5430 Centrifuge R, an Eppendorf 5920 RCentrifuge, a Jouan Robotics GR4 Auto Centrifuge, or a Thermo FisherScientific Heraeus Multifuge X3 Series Centrifuge. However, the presentdisclosure is not limited thereto.

Each multi-well plate has a corresponding counter balance 1814, whichensures the centrifuge 1818 is stable during operation. Without humanintervention, a mass of each counter balance 1814 is adjusted to beequal to the mass of the corresponding multi-well plate. This adjustmentis accomplished by pumping fluid (e.g., water, mineral oil, aqueoussolutions, organic solutions, etc.) from a container 1806, whichincludes a pressure regulator 1808, or in some embodiments an airintake, to the counter balance 1814 via a pump 1810. In someembodiments, the pump 1808 is a siphon. Attached to the pump is anozzle, which either dispenses fluid from the container 1806 to thecounter balance 1814 or draws fluid from the counter balance 1814 to thecontainer 1806. In some embodiments, a bottom end portion of eachcounter balance 1814 is formed with a slope to allow the for pooling ofthe fluid of the container 1806 in the counter balance 1814. The poolingof the fluid in the counter balance 1814 ensures that the entire fluidcan be completely removed from the counter balance. In some embodiments,the slope is formed as a V-shape, as a U-shape, or as a slant. Moreover,in some embodiments each counter balance 1814 is configured to have arelatively low empty mass (e.g., a few grams) to allow for higheraccuracy and precision in determining a mass of the counter balancewhile minimizing energy consumption required to move the counterbalance. Each counter balance holds a high internal volume or capacityto ensure that sufficient mass can be added therein, and a hightolerance to centrifugal action to ensure that the counter balancesurvives operating in the centrifuge.

In some embodiments, a multi-well plate and the corresponding counterbalance 1814 are disposed on a balance 1816, or scale, in order toensure that the masses of the multi-well plate and the correspondingcounter balance 1814 are equal. This also ensure that the measurementsare conducted simultaneously. Likewise, in some embodiments the mass ofthe multi-well plate is determined using the scale 1816 and stored forlater use. The multi-well plate is disposed into the centrifuge 1818 bythe robotic arm 1802. The mass of the corresponding counter balance isthen determined the scale 1816, and adjusted in accordance with thestored mass of the multi-well plate. In some embodiments, the mass ofthe corresponding counter balance is stored for later use, such that theadjustment of the mass of the counter balance in a later operation ofthe centrifuge does not require as much of an adjustment (e.g.,adjusting a previous mass of 20 g to 21 g instead of adjusting a mass of0 g to 21 g). Once the mass of the multi-well plate and the mass of thecorresponding counter balance 1814 are equal, and both devices aredisposed in the centrifuge 1818, the centrifuge is operated.

In some embodiments, after operating the centrifuge 1818 with the firstset of multi-well plates and corresponding counter balances 1814, asecond set of multi-well plates are prepared accordingly. In someembodiments, the second set of multi-well plates is the first set ofmulti-well plates, which were previously sterilized. A mass of eachmulti-well plate in this second set is determined, and masses ofcorresponding counter balances 1814, are determined in accordance withthe above descriptions of the first set of multi-well plates (e.g.,determined simultaneously or consecutively). Accordingly, the utilizedautomated centrifuge system of the present disclosure enables thecentrifuge 1818 to be fully operated (e.g., multi-well plates areproduced and masses of counter balances are determined and adjusted)without human intervention.

Workflows can include processes for pathway construction, expressionfine-tuning, genome editing, and cell adaptation but the presentdisclosure is not limited thereto. Other workflows include cloning,evolutionary organic engineering, genome organic engineering,genotyping, library screening, pathway construction, and protein organicengineering (1016).

Examples of process modules includes, but are not limited to, cellculture, DNA assembly, DNA purification, DNA quantification,normalization, polymerase chain reaction (PCR), sample preparation,sampling, sample analysis, protein extraction, and transformation;however, the present disclosure is not limited thereto. For instance, inother embodiments, such as a surgical pathology system or a toxicologysystem, process modules can various from system to system (1018, 1020).

Examples of unit operations include, but are not limited to,centrifugation, chilled incubation, heated incubation, magneticseparation, peeling, pipetting, dispensing, sealing, shaking incubation,spectrophotometry, chromatography, mass spectrometry, microscopicimagining, electrophoresis, electroporation, clone separation, colonyselection, and thermal cycling. Other unit operations include freezing,purifying, heating, cryogenic storage, sonication, milling, sterilizing,and the like (1022, 1024).

An instrument is a device that conducts a specific function or functionsin the automated system. In most cases, an instrument is a device thatconducts a unit operation or unit operations to samples or organicengineering targets. Examples of instruments include, but are notlimited to, a centrifuge, a Peltier temperature controller, anincubator, a shaking incubator, a magnetic separator, a peeler, a liquidhandling robot, a dispenser, a sealer, a plate reader, a liquidchromatography system, a gas chromatography system, a mass spectrometrysystem, a microscope, a electrophoresis device, a electroporationdevice, a clone separation device, a clone selection device, and athermal cycler. FIG. 6B depicts relations between exemplary unitoperations and a plurality of instruments. Other instruments include,but are not limited to, fume hoods, glove boxes, stability chambers,sterilizers, mills, burners, water baths, coolers, and similarinstruments which are found in scientific laboratories and biologicalfoundries (1026, 1028).

For instance, referring to FIG. 13, in some embodiments an instrumentincludes a reagent handling device 1300. The reagent handling device1300 of the present disclosure in configured to store and dispense avariety of reagents (e.g., liquids and/or gases reagents) for prolongedperiods of time. For instance, if a reagent is honey, which has aninfinite expiration date, the reagent handling device could store anddispense the honey as long as there is power supplied to the robot 1300.

The reagent handling device 1300 of FIG. 13 includes a primary switch1302 (e.g., a primary valve) which is configured as a master control ofthe robot. Control of the reagent handling device 1300 is conductedthrough one or more pumps (e.g., a multitude of individual pumps, amultichannel pump, or a combination thereof), that allows for control ofa dispensing cycle and a sterilization cycle. The primary switch 1302 isprogrammatically controlled to allow for automated operation of therobot 1300 in a workflow. However, in some embodiments, the primaryswitch 1302 is controlled manually and/or the primary switch 1302 is aplurality of selective valves. In some embodiments, the primary switch1302 includes at least a first port 1302-1 and a second port 1302-2,which are dedicated ports dedicated to various cycles of the reagenthandling device 1300, which will be described in more detail infra.

The dispensing cycle includes a dispense manifold 1310 and a reagentcontainer portion. An inlet of the dispense manifold 1310 is coupled toan outlet of the primary switch 1302, allowing reagents to flow ortraverse from the reagent container portion to the dispense manifold. Anoutlet of the dispense manifold 1310 is coupled to one or moredispensers 1312. The present illustration depicts six dispensers (e.g.,dispenser 1312-1, dispenser 1312-2, . . . , dispenser 1312-6). However,the present disclosure is not limited thereto. For instance, in someembodiments there exists a one-to-one relation between reagents anddispensers 1312 of the reagent handling device 1300. In someembodiments, there exists multiple dispensers 1312 in order to alloweach reagent and combinations thereof to be dispensed through adedicated dispenser 1312.

Furthermore, in the present illustration there is one connection fromthe primary switch to the dispense manifold. In some embodiments, thereare multiple connections from the primary switch to the dispensemanifold. Multiple connections for a dedicated connection to thedispense manifold 1302 for each reagent that the reagent handling device1300 is configured to dispense. Each coupling or connection of thereagent handling device 1300 is a tubing that allows for a sterile forof reagent therein. In some embodiments, these connections areremovable.

The reagent container portion includes a variety of reagent containers1304 (e.g., reagent container 1304-1, reagent container 1304-2, . . . ,reagent container 1304-6, . . . reagent container 1304-i), each storinga unique reagent. In some embodiments, the reagent containers are formedof a sterile material, are sealed from an external environment, areresistance to electromagnetic radiation (e.g., ultraviolet light,visible light, infrared light, etc.), or a combination thereof.Moreover, there is no maximum or minimum number of reagent containers1304 that are utilizable by the reagent handling device 1300, since therobot is configured to store and dispense any number of reagents.

In some embodiments, each reagent container 1304 is coupled to apressure regulator 1316. The pressure regulator 1316 allows for acontrol of pressure within the reagent container in order to eitherincrease a pressure of the reagent container or decrease the pressure.Control of pressure within the reagent container 1304 enablesdisplacement of an internal reagent with air, or another fluid. In someembodiments, the pressure regulator 1316 includes an air filter thatprevents contamination of the reagent container 1304. Air filters shouldbe appropriately selected in relation to prevent microbial andparticulate contaminants from entering the reagent container. In someembodiments, the pressure regulator 1316 includes a check valve, whichprevents evaporation and/or vapor release of a reagent in the reagentcontainer 1304. Furthermore, each reagent container 1304 is coupled to avalve 1314 (e.g., a selection valve), which controls a flow of materialto/from the reagent container and the primary valve 1302 or a washmanifold 1308.

The sterilization cycle includes the wash manifold 1308 and asterilization container 1306. The wash manifold 1308 controls a flow ofa sterilization fluid from the sterilization container 1306 to eachreagent container 1304. In some embodiments, the sterilization containeris a reagent container 1304 that is dedicated to storing thesterilization fluid. Sterilization fluids include liquid chemicalsterilants, disinfectants (e.g., high level disinfectants), distilledwater, detergents, and similar cleaning solutions such as a buffer. Insome embodiments, the sterilization fluid is a fluid which thermallysterilizes, such as steam. In some embodiments, the sterilization fluidis a low temperature gas, vapor, or plasma. The sterilization container1306 is configured in a similar manner as the reagent container 1306, inthat a valve 1314 and a pressure regulator 1316 are coupled thereto.

While describing the cycles of the liquid handling robot 1300, a firstposition of a valve 1314, hereinafter “P1,” refers to a valve position1314-P1 as shown and described by exemplary reagent container 1304-5 ofFIG. 13; a second position of a valve 1314, hereinafter “P2,” refers tovalve position 1314-P2 as shown and described by exemplary reagentcontainer 1304-5 of FIG. 13; a third position of a valve, hereinafter“P3,” refers to valve position 1314-P3 as shown and described byexemplary reagent container 1304-5 of FIG. 13.

In some embodiments, the sterilization cycle is a two-part cycle (e.g.,a first sterilization cycle and a second sterilization cycle). The firstcycle is configured to sterilize select portions of the reagent handlingdevice 1300 that require, or have previously required, attention (e.g.,repair or replacement), while the second cycle is configured tosterilize portions of the reagent handling device during automated useof the robot.

In some embodiments, the second cycle is configured to sterilize selectportions of the reagent handling device 1300 that were recentlydispensed or activated, and purge select portions of the reagenthandling device 1300 with air, gas, distilled water, or a similarmedium.

While describing the cycles of the liquid handling robot 1300, a firstposition of a valve 1314, hereinafter “P1,” refers to a valve position1314-P1 as shown and described by exemplary reagent container 1304-5 ofFIG. 13; a second position of a valve 1314, hereinafter “P2,” refers tovalve position 1314-P2 as shown and described by exemplary reagentcontainer 1304-5 of FIG. 13; a third position of a valve, hereinafter“P3,” refers to valve position 1314-P3 as shown and described byexemplary reagent container 1304-5 of FIG. 13.

The first sterilization cycle (e.g., an initial sterilization cycle), isconducted by selecting a portion of the reagent handling device 1300(e.g., reagent container 1304-5) to sterilize. The primary valve 1302 isswitched to the selected reagent container, the valve 1314 of theselected reagent container 1304 is switched to P1 and P2 (e.g., P1 open,P2 open, P3 closed), and the valve 1314 of the selection sterilizationcontainer 1306 to P2 and P3. This first sterilization cycle allows for,in part, various amounts of sterilization fluid to be dispensed to avariety of dispensers 1312 from the sterilization container 1306 throughthe wash manifold 1308 to the selected reagent containers 1304.

The second sterilization cycle (e.g., an automated sterilization cycle),is conducted by switching the primary valve 1302 to the second port1302-2. This second sterilization cycle allows for, in part, variousamounts of sterilization fluid to be dispensed to a variety ofdispensers 1312 from the sterilization container 1306 through the washmanifold 1308 to the selected dispensers 1304. To purge the primaryvalve 1302 and the dispenser 1312, the primary valve is switch to portone 1302-1, which allows gas or air to be pumped through portions of thesystem.

The dispensing cycle is conducted by switching each reagent container1304 to P1 and P3. The primary valve 1302 is switch to a selectedreagent container 1304, which allows a flow of material from theselected reagent container to a predetermined dispenser 1312.

Referring to FIG. 14, in some embodiments the instruments include acentrifuge compatible vacuum manifold 1400. The centrifuge compatiblevacuum manifold drives fluid through a resin or a filter column. Asillustrated in FIG. 14, in some embodiments the centrifuge vacuummanifold 1400 includes a plate gasket 1402, which seals a portion (e.g.,a top end portion) of the manifold 1400. The manifold 1400 also includesa channel plug 1404, which controls and prevents a flow of fluid out ofa container body 1406 of the manifold. The container body includes oneor more fluid channels formed therein, which guide a flow of fluidtowards the channel plug 1404. In some embodiments, the fluid channelsare formed with a declined slope on a bottom surface of the containerbody 1406 to assist a flow of fluid. The channels of the container body1406 are configured to have a first opening of the container body 1406be at a lowest point of the container body, to have at least a portionof one or more channels at a higher point than the lowest point of thecontainer body, and have a second opening at an outside portion of thecontainer body 1406 that attaches to an external vacuum. Furthermore, avacuum coupler gasket 1408 is coupled to the manifold 1400 to seal themanifold to the external vacuum, which rests in a vacuum nest 1410. Thevacuum next is removably coupled to the manifold 1400, and supports, orcouples to the external vacuum. In some embodiments, the manifold 1400is configured to have a footprint in accordance with guidelines of theSociety for Biomolecular Screening (SBS). ANSI SLAS 1-2004 (R2012)., thecontents of which are hereby expressly incorporated by reference.Moreover, in some embodiments the manifold 1400 is configured to have alow profile in order to fit the manifold into a variety of centrifugesand dispensers. The low profile allows the manifold 1400 to couple tocentrifuges which may have column arrays attached to thereto. In someembodiments, the vacuum gasket 1408 and the vacuum nest 1410 havechamfered and/or filleted boundary portions to enable a fit andpositioning of the container body 1406. As previously described,chamfered and/or filled boundary portions of instruments and componentsthereof is preferred in order to allow an automated robot to fit trays,multi-well plates, samples, and the like into instruments withoutyielding a hard collision. For instance, if a tray is misallied by 0.1mm, and a fillet has a radius larger than this misalignment, than thetray will follow the curve of the fillet and fit into a receptacle.Furthermore, in some embodiments the vacuum gasket 1408 and the vacuumnest 1410 are removably coupled to the container body 1406. In someembodiments, each component of the manifold 1400 is removably coupled,such that the entire manifold can be disassembled into its constituentcomponents. Accordingly, the centrifuge compatible vacuum manifold 1400of the present disclosure is operable by a robot arm (e.g., robotic arm1802 of FIG. 18, robotic arm 1902 of FIG. 19), acts as a vacuummanifold, and stores any fluid dripped from a column array until avacuum is applied.

Referring to FIGS. 15 through 17, in some embodiments the instrumentsinclude a clone separate apparatus, which allows for an automatedmicrobial culture generation. As illustrated in FIG. 15, a pipette 1500is utilized to aspirate distilled water 1502, or a similar buffer ordiluent medium, and air 1504, or a similar gas or medium, in analternating manner. These alternative aspirations are repeated apredetermined number of times, according to a design of the presentdisclosure. For instance, in some embodiments, there are five totalalternations, ten total alternations, twenty total alternations, orfifty total alternations. In some embodiments, a volume of eachaspiration is uniform (e.g., a volume of aspiration 1502-1 is equal to avolume of aspiration 1504-1, which is equal to a volume of aspiration1502-2, etc.). In some embodiments, a volume of each type of aspirationis uniform (e.g., a volume of aspiration 1502-1 is equal to a volume ofaspiration 1502-2, which is equal to a volume of aspiration 1502-3,etc.). Furthermore, in some embodiments a volume of each aspiration isnon-uniform (e.g., a volume of aspiration 1502-1 is not equal to avolume of aspiration 1504-1 or a volume of aspiration 1502-2, which isnot equal to a volume of aspiration 1502-3, etc.) Once the finalaspiration of air 1504-i is drawn, a final portion of diluent 1502-Z isaspirated. However, the present invention is not limited thereto, as insome embodiments the final aspiration 1502-Z is omitted. Finally, a cellculture 1506 is aspirated.

Referring to FIG. 16, once the contents of the pipette 1500 have beenfull aspirated, the contents are gradually dispensed into a cell culturemedium 1602 (e.g., a petri dish). The dispensing direction is indicatedby an arrow 1604 of FIG. 16A and FIG. 16B. In some embodiments, thedispensing direction is a line, a predetermined curve (e.g., a sinecurve, a parabolic, a cubic function, etc.), or series of curves (e.g.,a parabolic function coupled to a linear line). During dispensing of thecontents of the pipette 1500, diluent 1502, air 1504, and the cellculture 1506 exhibit plug flow. However, the present disclosure is notlimited thereto, as one skilled in the art may which to exhibit otherflow types during dispensing of the contents of the pipette 1500. Insome embodiments, a density of contents dispensed decreases as thecontents are dispensed, such that an initial dispensing area has a highdensity while a final dispensing area has a low density. Similarly, insome embodiments the initial dispensing area has a lower density whilethe final dispensing area has a high density.

Referring to FIG. 17, a streaking cone 1700 is coupled to either thepipette 1500 or a similar device through a coupling portion 1706 of thestreaking cone. In some embodiments, the coupling portion 1706 includesa cavity 1708 configured to receiving the pipette 1500. The streakingcone 1700, or a device coupled thereto (e.g., the pipette 1500), is thenmoved in a continuous motion from a first position to a second position.In some embodiments, the streaking cone is moved in a first continuousmotion from a first position to a second position, then in a secondcontinuous motion from a third position to a fourth position. FIGS. 16Band 16D illustrate various spiral continuous motions of the streakingcone 1700 according to embodiments of the present disclosure. However,the present invention is not limited thereto. For instance, in someembodiments, the streaking is performed in a concentric pattern (e.g.,concentric circles, concentric rectangles, concentric triangles) or inan array (e.g., an array of lines, an array of circles, etc.) Thecontinuous motion of the streaking cone is configured to spread thedispensed contents of the pipette 1500 using a tip 1702 of the streakingcone. The tip 1702 of the streaking cone can be designed in a variety ofshapes which promote various spreads of cell cultures. Furthermore, insome embodiments the streaking cone 1700 includes an elastic, orflexible, portion 1704 which allows for motion in a vertical axis of thestreaking cone. In the present illustration, the flexible portion 1704is formed as type of spring. However, in some embodiments, the flexibleportion 1704 is formed as a damper or similar energy absorbing device.Accordingly, the clone separation apparatus of the present disclosureenables a full range of dilution ratios are realized, which guaranteesgeneration of multiple single colonies.

In some embodiments, the first device is in electronic communicationwith at least one transport path coupled to the plurality of instrumentsfor receiving a sample from the plurality of instruments and returningthe sample to the plurality of instruments. The transport path isutilized to transfer a sample or organic engineering target from aninstrument, a unit operation, or a process module to another instrument,unit operation, and/or process module. In many embodiments, thetransport path allows a given sample or organic engineering target totraverse three dimensions in a laboratory without human input. In manyembodiments, the transport path is the free volume in the system which atransporter can operation unobstructed. The transport path can comprisea multi-lane path or a grid of paths (1030).

In many embodiments, the transport path comprises at least onetransporter configured to move about the transport path. A physicalstorage medium, or buffer, is disposed on the at least one transporter.The buffer is configured to hold a sample or an organic engineeringtarget temporarily to free, or allow, additional operations in thesystem. For instance, when a sample needs to be disposed in anincubator, but said incubator is occupied for 10 minutes and theinstrument said sample was previously disposed requires immediate use byanother second sample, the original sample can be disposed in the buffertemporarily to free instruments or arms of a transporter for furtheruse. In many embodiments, the at least one transporter comprises arobotic arm, a ground vehicle, a reduced friction ortho-multilaneconduit, a drone, a conveyor belt, a transfer station, a lift, a crane,an elevator or a combination thereof. The present disclosure is notlimited thereto, and many types of transportation devices can beutilized by a person skilled in the art of the present disclosure. Inmany embodiments, the at least one transporter comprises a liquidhandling robot. In such embodiments, a typical transportersconfiguration requires a first transporter as a general transporter anda second transport as a liquid handling robot. Transporters can utilizeseries or parallel routes, as well as combination routing of series andparallel processing routes. These combinations can reduce travellingtimes through optimized flexible routing between unit operations and/orinstruments. Additionally, the transporter and transport path should beconfigured to reduce friction, thus minimizing operating forces, toincrease smoothness of transportation. This decrease a risk of sampledropping, spilling, cross contamination and the like. (1032, 1034,1036).

The method further requires translating, for each respective organicengineering target in the first plurality of organic engineeringtargets, the first uncompiled workflow into a corresponding instance ofa compiled first workflow for the respective organic engineering target.In general, an uncompiled workflow is a workflow prior to obtaining aparticular sample input. An instance of a compiled workflow is a singleiteration of a workflow among a plurality of iterations of said workflow(1038).

In some embodiments, such as biofoundry, each respective compiledworkflow in the plurality of instances of the compiled first workflow isa scheme to synthesize and express a pair of TALEN in a singletranscript format by a P2A self-cleavage sequence (1040).

In such embodiments, at least 400 pairs of TALENs are expressed in a24-hour time interval; however, the present disclosure is not limitedthereto. In some embodiments, at least 200 pairs of TALENs are expressedin a 24-hour time interval, and in another embodiment at least 600 pairsof TALENs are expressed in a 24-hour time interval. An exact number ofcompleted workflows or expressed organic engineering targets may varydepending on a number of environment, workflow, and system conditions(1042).

In some embodiments, the first organic engineering target in the firstplurality of organic engineering targets is converted into one or morefirst inputs for the first uncompiled workflow. For instance, when thefirst organic engineering target is an end goal or desired output, suchas salt (NaCl), the engineering target is converted into the reagentcomponents of that output, such as Sodium (Na) and Chlorine (Cl) (1044).

In some embodiments, the first organic engineering target is a synthesisof a first nucleic acid and the one or more first inputs for the firstuncompiled workflow are a set of nucleic acid bases for synthesizing thefirst nucleic acid (1046).

In some embodiments, the first uncompiled workflow includes a branchcondition, a loop condition, or nested condition, and the translatingresolves the branch condition, loop condition or nested condition basedon a value associated with the branch condition, loop condition ornested condition in order to form the linear temporal order of the firstplurality of unit operations. In some embodiments, loop conditions arelogical criterion to exit a loop. In some embodiments, branch conditionsare logical criterion to determine the branch for the program to proceedat a fork. The logical conditions usually require the input from theexperiment or workflow itself. For example, after running a sample batchin Process Module A, when all measurements surpass threshold X, thenexecute Branch N. (1048).

Each respective instrument in the first subset of instruments includesan address of the respective instrument and one or more executioninstructions for the respective instrument. Instrument executioninstruction(s) are at least parameter sets used in programming of unitoperations, process modules, and workflows. The instrument executioninstructions sets are the configurations of machines and processconditions. Such machine configurations and process conditions include,but are not limited to, adjusting rotations per minute (RPM) of amachine, changing a status of a machine from or to ON and OFF, and thelike. Another interpretation of instrument execution instructions is alogical dependency of unit operations in a workflow that defines theprocedures that samples are processed (1050).

For example, an instrument executable instruction(s) can include, butare not limited to:

run Unit Operation 1 with Parameter Set 1 for Sample IDs A-Z

run Process module 1 with Parameter Set 2 for Sample IDs A-Z

if Results from Process module 1≥Threshold 1, then

run Process module 2 with Parameter Set 3 for Sample IDs A-Z

else if

run Process module 3 with Parameter Set 4 for Sample IDs A-Z

Instrument execution instructions can either be interpreted as specificvalue or coordinate instructions such as:

Parameter Set 1 through n

Sample 1 through n

Threshold 1 through n

Additionally, instrument execution instructions can be interpreted asdependencies in the process such as:

execute 2) after 1)

execute 3) after 2)

execute 4) after logic decision 3)

execute 6) after logic decision 3

In some embodiments, the address of the respective instrument comprisesspatial coordinates including, but not limited to, Cartesiancoordinates, polar coordinates, spherical coordinates, jointcoordinates, or tool coordinates of the respective instrument. In someembodiments, the address of the respective instrument comprises aphysical location of the respective instrument. In many embodiments, theaddress of the respective instrument comprises a unique electronicaddress of the respective instrument such that the first device, such ascomputer 10, can communicate with the instruments electronically. Thecorresponding instance of the respective compiled workflow furthercomprises an operating condition for the respective instruction. Theoperating condition of the respective instruction can include aparameter of an instruction such as a final value check or initialverification. (1052, 1054, 1056, 1058).

Referring to FIG. 19, positioning and control methods and systems ofvarious instruments will be described in relation to a sample foundry1900. The foundry 1900 includes a transporter 1902, a variety ofinstruments 1904, and a variety of markers 1906. In some embodiments,the transporter 1902 includes an articulated handling robot, orextension, which handles various samples and subcomponents of theinstruments 1904. In some embodiments, the articulated handling robot isthe handling robot 1802 of FIG. 18.

Each instrument 1904 of the foundry 1900 includes a respective address,which is a coarse grain address, that is identified through acorresponding marker 1906. Coarse grain addresses allow the transporter1902 to position itself in a position suitable to operate a respectiveinstrument within a tolerance of from 1 millimeter (mm) to 20 mm.Markers 1906 provide positional information to localize the transporter1902. Accordingly, the positioning system locates and guides thetransporter 1902 to a position proximate to the respective instrument1904 of the marker 1906 using the coarse grain address.

In some embodiments, instruments 1904 and corresponding markers 1906exist in a one-to-one relation (e.g., instrument 1904-1 is unique tomarker 1906-1), a many-to-one relationship (e.g., instruments 1904-3 and1904-4 are associated with marker 1906-2), or a one-to-many relationship(e.g., instrument 1904-2 is associated with markers 1906-3 through1906-6 which correspond to different orientations of the instrument). Insome embodiments, when an address, or position, of a marker 1906 isdetermined, the address is stored for later user. For instance, when aposition of a first marker is detected, the detected position is storedsuch that when the first marker is recalled upon in a later instance ofa workflow, the position of the marker does not need to be redetected.In some embodiments, the position is stored in non-volatile memory. Insome embodiments, the position is stored in volatile memory.Accordingly, the position of a marker is re-determined for eachworkflow.

In some embodiments, the positioning system is a global positioningsystem (GPS) and each marker 1906 is a GPS coordinate. Likewise, in someembodiments the positioning system is a radio frequency (RF) system andeach marker 1906 is a RF signal generator. Moreover, in some embodimentseach marker 1906 is a two-dimensional (2D) marker (e.g., a barcode, aquick-reference (QR) code, a color patch, etc.), which is detected andcaptured using a variety of sensors (e.g., light detection and ranging(LIDAR) sensors, ultrasonic sensors, encoders, a compass, etc.) or acamera (e.g., a high resolution camera such as the JS 1080p, 120degrees, distortion free camera or RerVision USB8MP02G camera) attachedto the transporter 1902. These sensors and/or camera feed real timeinformation to a controller and provide information related to a spatiallocation of the transporter and/or articulated handling robot of thetransporter, as well as their surrounding environment. For instance, insome embodiments, the transporter includes one or more LIDAR sensors ordetectors which use electromagnetic waves to detect obstacles in athree-dimensional environment (e.g., a biological foundry). In someembodiments, the LIDAR sensor is a SICK LMS-111 LIDAR sensor. This LIDARsensor operate at a wavelength of 905 nanometers (nm) (e.g., infrared),with an aperture angle of 270° and a scanning frequency of 25 Hertz (Hz)to 50 Hz. However, the present disclosure is not limited thereto. Forinstance, in some embodiments the wavelength of light used by a sensoris of from 10 micrometers to 250 nm.

In some embodiments, the positioning system is a set of predeterminedpaths. An inertial measurement unit (IMU) sensor is coupled to thetransporter 1902 in order to detect disturbances to the transporter andcorrect for the disturbances. The IMU sensor includes an accelerometer,a gyroscope, and a magnetometer. When a disturbance is detected throughthe IMU sensor, the transporter 1902 adjusts its position to accord forthe disturbance and remain in a stationary position, or remain on itscurrent transport path. In some embodiments, the IMU sensor is a LordSensing 3DM-GX5-25 IMU sensor. Furthermore, in some embodiments thepositioning system includes a series of magnetic scales, where themarkers 1906 are magnetic markers. Accordingly, the transporter 1902traverses the magnetic scales and locates the instruments through themagnetic makers 1906. In some embodiments, the sensors of the presentdisclosure have a suitable range such that all instruments of a foundryare within the range of the sensor.

In some embodiments, the above described positioning systems areutilized to form a map, or representation, of a surrounding environmentof the foundry 1900 in order to generate predetermined paths for thetransporter 1902. In some embodiments, the above described positioningsystems are utilized to form a map, or representation, of a markerpositioning in the foundry 1900 in order to generate predetermined pathsfor the transporter 1902. Furthermore, in some embodiments, a Gaussianblurring function or low-pass filter is applied to either therepresentation or captured feed to de-noise an image and limit a rangeof error in determining a positioning of each marker 1906. In someembodiments, the representation is formed such that the transporter 1902is an origin, or zero, of the representation. Similarly, in someembodiments the representation is formed such that a center of asurrounding environment of the transporter 1902 is an origin, or zero,of the representation.

In accordance with a determination that the transporter is at a correctcoarse grain address (e.g., marker 1906) of a respective instrument1904, the transporter is held in position. In some embodiments, thetransporter is held in position by being lifted through one or moreextending linear actuators. Similarly, in some embodiments thetransporter is held in position through feedback of aproportional-integral-derivative loop controller in communication withthe sensors.

In some embodiments, while the transporter 1902 is actively moving, thesensors (e.g., LIDAR sensor and/or IMU sensor) are active in detectingthe surrounding environment of the transporter and marker positions.Accordingly, the transporter 1902 is capable of reacting to adisturbance in its transport path (e.g., detecting a person or obstaclemoving through, or standing in, the transport path). This reactionallows the transporter to slow down, stop, or reroute to a differenttransport path depending on each disturbance and workflow.

The articulated hand utilizes a fine transverse instruction to move toat least one spatial coordinate of a respective instrument. Forinstance, in some embodiments a fine traverse instruction is a commandto extend the articulated arm in a horizontal line.

In some embodiments, while handling various samples the articulatedhandling robot transfers a tray (or similar component of an instrumentor workflow such as a vial or container) to an instrument 1904 using asafety transfer procedure, which is type of fine traverse instruction.This safety transfer procedure is conducted when a readout from thesensors, or similar positioning system, fails to satisfy a thresholdproximity location (e.g., a threshold of 1 mm, a threshold of 5 mm, athreshold of 20 mm, a threshold of 100 mm, a threshold of 0.1 degrees'rotation, a threshold of 0.25 degrees' rotation, a threshold of 0.5degree's rotation, a threshold of 1 degree rotation, etc.) of an actuallocation of the instrument. Similarly, when the threshold proximitylocation is satisfied, a default transfer procedure is conducted. Forinstance, in some embodiments the transporter 1902 determines that it isin a correct positioning in relation to a corresponding marker 1906.However, the sensors detect that the transporter 1902 is in an incorrectposition, which enables the safety transfer procedure.

In some embodiments, the safety transfer procedure includes utilizing aforce detection sensor, which allows for compliance in the motion of therobot. In some embodiments, the force detection sensor is a torquesensor. The safety transfer procedure initiates a small (e.g., tightlygrouped), slow oscillating motion to gradually load a container to areceiving portion of an instrument. The oscillating motion includes asimple oscillation, a Lissajous oscillation, or a spiral-shapedoscillation. For instance, in some embodiments the articulated armextends in a direction, with small (e.g., 2 mm radius) spirals orientedabout the direction of extension. When a force is detected by the forcesensor, the path of extension is modification to be compliant with thedetected force. Since the robot is compliant to detected forces anddisturbances, the oscillating motion is constrained to the boundariesand chafers of the instrument, which allows for the articulated handlingrobot to find and fit the container into the instrument without a hardcollision caused by incorrect positioning.

The method further requires the first plurality of unit operations to betemporally organized into a linear temporal order. Each respective unitoperation in the first plurality of unit operations is characterized bythe time interval of the corresponding unit operation definition. Forexample, referring to FIG. 6A, process module DNA Quantificationincludes unit operations Spectrophotometry and Pipetting. The unitoperation definition(s) for Spectrophotometry defines a 40-minute timeinterval, and the unit operation definition(s) for Pipetting defines a10-minute time interval. Thus, the first plurality of unit operationswould have required at least 50 minutes and have at least two plausibleworkflows. As such, a plurality of instances of the compiled firstworkflow are formed (1060).

In some embodiments, the method enables the user of the first device,via a graphical user interface or otherwise, to adjust the lineartemporal order of the first plurality of unit operations. Such graphicaluser interfaces include, but are not limited to, the Gantt chartdepicted in FIG. 10. In FIG. 10, “Batch 1” refers to a first workflowand “Batch 2” refers to a second workflow. A user of the first devicecan adjust and order the unit operations of a workflow of at theirdiscretion. In some embodiments, predetermined contingency checks mayprevent a user from ordering unit operations in less than optimalconfigurations or configurations which trigger a predetermined alert(1062).

In some embodiments, each organic engineering target in the firstplurality of organic engineering targets is an input into acorresponding instance of a compiled first workflow in the plurality ofinstances of the compiled first workflow.

In some alternative embodiments, each organic engineering target in thefirst plurality of organic engineering targets is an output into acorresponding instance of a compiled first workflow in the plurality ofinstances of the compiled first workflow.

In general, an engineering target can be, at any given point in time, aninput or output of an instance of the same compiled workflow. Likewise,an input of a workflow can be the input of another workflow (1064,1066).

In some embodiments, the method further requires obtaining, via the oneor more peripheral devices, a second plurality of organic engineeringtargets. As previously described, the second plurality of engineeringtargets or samples can exist is a variety of forms, including, but notlimited to, the forms of the first plurality of organic engineeringtargets (1070).

In some embodiments, the second plurality of organic engineering targetsare determined from outputs of the plurality of instances of thecompiled first workflow. In such embodiments, the second workflow cancommence subsequent completion of the compiled first workflow (1072).

Furthermore, in some embodiments, the method assigns the secondplurality of organic engineering targets to a second uncompiledworkflow. Like the first uncompiled workflow, the second uncompiledworkflow is configured to produce the second plurality organicengineering targets, and the second uncompiled workflow is associatedwith a second subset of process modules in the plurality of processmodules (1074).

In some embodiments, the method further performs a second translating,for each respective organic engineering target in the second pluralityof organic engineering targets, the second uncompiled workflow into acorresponding instance of a compiled second workflow for the respectiveorganic engineering target (1076).

In many embodiments, two or more instances of the compiled firstworkflow are executing at a time when the second translating isexecuted. (1078) As used here, a compiled workflow is “executing” whenat least one unit operation of the compiled workflow is presently beingserviced by an instrument specified by the unit operation. For example,consider the case where a unit operation in a compiled workflowspecifies that an aliquot of fluid be pipetted into a tube. In someembodiments, the compiled workflow that contains unit operation is“executing” during the actual physical pipetting operation specified bythe unit operation while the instrument is performing the pipetting asinstructed by the unit operation. In some embodiments, the compiledworkflow that contains a unit operation is “executing” during the entiretime interval in the unit operation that contains this pipettingoperation, not just the actual physical amount of time that it takes theinstrument to perform the pipetting. Thus, in such embodiments, thecompiled workflow is deemed to be “executing” across the entire timeinterval of the unit operation, even if the physical instructions of theunit operation are completed by the specified instrument before theentire time interval is completed. More generally, a compiled workflowis deemed to be executing in some embodiments when the executioninstructions of any respective unit operation of the compiled workflowis currently controlling an instrument in the plurality of instrumentswithin the time interval specified by the respective unit operation.

Each respective instrument in the second subset of instruments includesan address of the respective instrument and one or more executioninstructions for the respective instrument. Like the instruments of thefirst subset of instruments, the respective addresses can exist is aplurality of forms including physical addresses and unique electronicaddresses (1080).

The second plurality of unit operations is temporally organized into alinear temporal order. Each respective unit operation in the secondplurality of unit operations is characterized by the time interval ofthe corresponding unit operation definition. Like the linear temporalorder of the first plurality of unit operations, the second plurality ofunit operations can be manipulated, via a graphical interface, by a userof the device or computer. (1082).

In some embodiments, two or more of the plurality of instances of thecompiled first workflow and two or more of the plurality of instances ofthe compiled second workflow are concurrently executed. As used herein,a “concurrently running” element refers to a unit operation of aworkflow is being currently enacted on an instrument in a plurality ofinstruments (1084).

In some embodiments, the first subset of instruments comprises two ormore instruments, the second subset of instruments comprises two or moreinstruments, and at least one instrument in the first subset ofinstruments is in the second subset of instruments (1086).

In some embodiments, the method requires concurrently executing three ormore of the plurality of instances of the compiled first workflow andthree or more of the plurality of instances of the compiled secondworkflow, wherein, the first subset of instruments comprises three ormore instruments, the second subset of instruments comprises three ormore instruments, and at least two instruments in the first subset ofinstruments is in the second subset of instruments. (1088)

In some embodiments, the method requires validating the second pluralityof unit operations according to a predetermined validation list. Thepredetermined validation list comprises one or more criteria of thecompiled second workflow. The one or more criteria of the compiledsecond workflow comprises a priority of each unit operation in thesecond plurality of unit operations, a weight of each unit operation inthe second plurality of unit operations, a time of completion for thesecond plurality of unit operations, a compatibility of the secondplurality of unit operations to a different plurality of unitoperations, a property of each unit operation in the second plurality ofunit operations, and one or more constraints of the second plurality ofunit operations. The property of each unit operation in the secondplurality of unit operations is selected from the set: a viscosityvalue, a purity value, a composition value, a temperature value, aweight value, a mass value, and a volume value. (1090, 1092, 1094)

In some embodiments, the method requires concurrently executing one ormore instances of the compiled first workflow and one or more instancesof the compiled second workflow, concurrently executing two or moreinstances of the compiled first workflow and three or more instances ofthe compiled second workflow, or concurrently executing three or moreinstances of the compiled first workflow and three or more instances ofthe compiled second workflow (1096, 1098, 1100).

In some embodiments, the method requires, at each respective time stepin a recurring series of time steps, simulating a remainder of each ofthe one or more instances of the compiled first workflow. This forms oneor more first simulations, each simulating a remainder of each of theone or more instances of the compiled second workflow, thus forming oneor more second simulations. Simulating a remainder of the one or moreinstances of a compiled workflow allows greater optimization in realtime and allows adaptation to new inputs and completed workflows. Aninterlocking condition error handler associated with a first unitoperation in an instance of the one or more instances of the compiledfirst workflow is fired which forms an interlocking condition with asecond unit operation in an instance of the one or more instances of thecompiled second workflow. (1102)

In some embodiments, firing the interlocking condition error handleradjusts one or more time intervals of one or more unit operations in aninstance of the compiled first workflow or an instance of the compiledsecond workflow that have not been executed. An interlocking conditionis a logical conflict in scheduling when one action require resourcesthat is being occupied by another action but can only be released whenthe first action proceeds. Firing the interlocking condition errorhandler can adjust various parameters, including, but not limited to, aweight one or more unit operations in an instance of the compiled firstworkflow or an instance of the compiled second workflow that have notbeen executed as a function of a priority assigned to the compiled firstworkflow versus a priority assigned to the compiled second workflow, oneor more time intervals of one or more unit operations in an instance ofthe compiled first workflow or an instance of the compiled secondworkflow that have not been executed as a function of a priorityassigned to the compiled first workflow versus a priority assigned tothe compiled second workflow, or an instance of the compiled firstworkflow or an instance of the compiled second workflow. In someembodiments, the interlocking condition error handler is a mutualexclusion error handler. The interlocking condition error handler canalso include a race condition or a lock condition (1104, 1106, 1108,1110, 1112)

In some embodiments, firing the interlocking condition error handlersuspends an instance of the compiled first workflow or an instance ofthe compiled second workflow. Suspending a workflow, as used herein,means aborting or ending a workflow or temporarily halting a workflow(1114).

In some embodiments, each time step in the recurring series of timesteps occurs on a periodic basis. In some embodiments, each time step inthe recurring series of time steps occurs every five minutes. In furtherembodiments, each time step in the recurring series of time steps occursevery 10 minutes, every 15 minutes, every 25 minutes, every 30 minutes,every 45 minutes, every 60 minutes, every 120 minutes, every half day,every day or the like (1116, 1118).

In some embodiments, each time step in the recurring series of timesteps occurs responsive to an occurrence of event in a plurality ofevent classes. An event class is an event triggered reschedulingcondition. This describes one type of rescheduling conditions that aretriggered by events such as equipment malfunction. Other such eventsinclude, but are not limited to, adding a new compiled workflow,instances of compiled workflows finishing with a delay or advance, whenactual decisions or looping cycles are not included in the simulatedinstances or workflows, abnormal resource status, such as malfunction,user interruption, instrument error, a power failure, a sample dropping,or an interlocking condition and the like. A rescheduling condition is alogical criterion for a rescheduling routine to be executed (1120,1122).

In many embodiments, the first subset of instruments comprises two ormore different instrument classes, and the second subset of instrumentscomprises two or more different instrument classes. Typically, aninstrument class can refer to a type of instrument, such as thepreviously mentioned 96 well and 24 well plates (1124).

A first instrument class and a second instrument class is used by boththe plurality of instances of the compiled first workflow and theplurality of instances of the compiled second workflow. The firstinstrument class has a first multiplex value and the second instrumentclass has a second multiplex value other than the first multiplex value.The method enacts a scheduler that maximizes a number of instances ofthe plurality of instances of the compiled first workflow, a number ofinstances of the plurality of instances of the compiled second workflow,or a number of instances of a combination of instances of the compiledfirst workflow and the compiled second workflow that can concurrentlyuse instruments of the first instrument class and instruments of thesecond instrument class given the first multiplex value and the secondmultiplex value. The scheduler orchestrates the unit operations on acontainer level, while a script generator (to be described infra)handles individual samples in said container in a pipetting operation.As such, the script generator converts experimental designs, such as DNAconstruct design, enzyme assay design, and/or restriction digestiondesign, into instrument executable instructions. The scripts generatedby the by the script generator will be utilized as a part of aconfiguration for unit operations when the scheduler dictates aworkflow. Additionally, classes of instruments can be utilized wheninstruments exist in various multiplex. For instance, when well platesexist in 96 well implementations and 24 well implementations, a firstsubclass includes each 96 well plate having a first multiplex value of 1and a second subclass includes each 24 well plate having a secondmultiplex value of 4. The multiplex values are typically utilized wheninstruments exist is various configurations and throughput of aplurality of devices needs to be optimized (1126).

The scheduler maximizes, at least in part, by invoking a first number ofinstances of the first instrument class as a function of the firstmultiplex value of the first instrument class and invoking a secondnumber of instances of the second instrument class as a function of thesecond multiplex value of the second instrument class to be runconcurrently support concurrently running instances of the compiledfirst workflow and the compiled second workflow (1128).

The scheduler maximizes, at least in part, by concurrently running afirst number of instances of the first compiled workflow and a secondnumber of instances of the second compiled workflow (1130).

The scheduler maximizes, at least in part, by adjusting, by an amount, atime interval of a respective unit operation in the first plurality ofunit operations of an instance of the first compiled workflow from thetime interval of the corresponding unit operation definition or byadjusting, by an amount, a time interval of a respective unit operationin the second plurality of unit operations of an instance of the secondcompiled workflow from the time interval of the corresponding unitoperation definition (1132).

Incorporated by reference in the present document is “Chao et al., 2017,“Fully Automated One-Step Synthesis of Single-Transcript TALEN PairsUsing a Biological Foundry,” ACS Synth Biol, 6, p 678”.

Example I—Design of a Single-Transcript TALEN Synthesis Scheme

The TALEN architecture used in this work is based upon the AvrXa10 TALEfrom Xanthomonas oryzae pv. oryzae as previously reported (Liang et al.,2014, “FairyTALE: A high-throughput TAL effector synthesis platform,”ACS Synth Biol, 3 (2), p 67). In brief, it utilizes a +207 aa N-terminusextension and a +63 aa C-terminus extension, which negates the 5′-Trequirement and allows greater flexibility in target sequence design(Sun et al., 212, “Optimized TAL effector nucleases (TALENs) for use intreatment of sickle cell disease,” Mol Biosyst, 8 (4), p 1255). Attachedat the C-terminus is an engineered FokI cleavage domain that showedgreater cleavage efficiency in yeast as well as human cells (Sun et al.,2014, “SunnyTALEN: a second-generation TALEN system for human genomeediting,” Biotechnol Bioeng, 111 (4), p 683). The central repeat domainsof the two TALENs are constructed from a library of di-repeatsubstrates, i.e., each substrate contains two TALE repeats thatrecognize two consecutive DNA bases. For this work, we used a library of441 di-repeat substrates, adapted from “FairyTALE”, equally divided into17 groups according to their position in the assembly (Liang et al.,2014, “FairyTALE: A high-throughput TAL effector synthesis platform,”ACS Synth Biol, 3 (2), p 67) (FIG. 11). In addition to the 4×4substrates to cover all possible DNA di-bases at each assembly position,we included the option to use either NH or NN to code for guanine“organic engineering targets”. To separate the two TALENs on the singleplasmid, we employed a poly-cistronic format utilizing a P2Aself-cleaving peptide sequence (Donnelly et al., 2004, “Multiple geneproducts from a single vector: ‘self-cleaving’ 2A peptides,” Gene Ther,11 (23), p 1673; Kim et al., 2011, “High Cleavage Efficiency of a 2APeptide Derived from Porcine Teschovirus-1 in Human Cell Lines,Zebrafish and Mice,” Plos One, 6 (4)). Both TALENs are coded as a singletranscript, but during translation, the P2A peptide will self-cleave thegrowing polypeptide to give two independent TALENs (FIG. 3A).

Using the set of optimized 4-bp junctions in the “fairyTALE”construction scheme, 2 sets of 7 di-repeats substrates, with a P2Alinker substrate in between, were ligated onto a TALEN receiver vectorin a single step via Golden Gate assembly. The N-terminus extension ofthe first TALEN and the C-terminus extension of the second TALEN werecarried by the vector, whereas the C-terminus extension of the firstTALEN and the N-terminus extension of the second TALEN were carried bythe linker substrate. Since the linker substrate and the receivercarried the last repeat of the two TALENs, 4 TALEN receivers and 4linker substrates were created (FIG. 4B). This construction schemeassembled 15 DNA fragments onto a 5 kb mammalian expression vector tocreate a single-plasmid TALEN pair that recognized a 30 bp DNA sequence.

Example II—One Pot Assembly of TALENs

To fulfill the requirements of TALEN library creation, we optimized thereaction condition to maximize the assembly fidelity. For librarycreation applications, picking individual clones for verification wouldbe an obvious throughput bottleneck, and we would therefore need toachieve high assembly fidelity to allow us to skip clonal isolationwithout drastically affecting the quality of the library. We picked 28colonies from a single-transcript TALEN assembly “organic engineeringtargets”, and assessed them by restriction digest followed by gelelectrophoreses. As shown in FIG. 4C, all 28 clones gave the correctdigestion pattern. We then sequenced 4 of the clones and they allappeared to be correct. This (28/28) corresponds to a fidelity of atleast 87.7% based on binomial probability with 95% confidence.

Example III—Single-Transcript TALEN Functionality in HEK293T and hESCCells

To ensure that P2A cleaves the protein effectively, we performed awestern blot analysis from the cell lysates of HEK293T that had beentransfected with single-plasmid TALENs. As shown in FIG. 5A, only TALENmonomer was detected and no dimer could be observed, suggesting that theP2A sequence cleaved the protein effectively in HEK293T cells.

After confirming P2A functionality, we went on to compare the DNAcleavage efficiency of single-transcript TALENs against previouslyreported traditional two-plasmid TALENs. Two sites, ABL1 and BRCA2“organic engineering targets”, were chosen for this comparison, and theexperiments “compiled workflows” were performed in HEK293T cells.Cleavage efficiency was measured using the T7E1 nuclease assay, whichdetects indels introduced via NHEJ after TALEN induced double strandedbreaks. As shown in FIG. 5B, the cleavage efficiency of the twosingle-transcript TALENs was comparable to that of traditional TALENs.The 1P-TALEN used in this experiment used NH to recognize guanine,whereas the traditional TALENs used NN to recognize guanine. Accordingto our observation and in agreement with that reported by others(Streubel et al., 2012, “TAL effector RVD specificities andefficiencies,” Nat Biotechnol, 30 (7), p 593), when used in largenumber, NH RVD is detrimental to TALE binding. We therefore recommendusing NN or a mix of NN and NH RVD when there are more than 4 guaninebases in the recognition site (FIG. 8A).

We further compared the cleavage efficiency of single-transcript TALENsin H1 hESC cells that had an IRES-EGFP marker behind the endogenous Oct4(H1 Oct4-EGFP, WiCell). We targeted OREG1393087 “organic engineeringtarget”, a site that is known to be an important enhancer for Oct4expression, with either traditional two-plasmid TALEN orsingle-transcript TALEN “organic engineering target”, and monitored theOct4 expression level in the stem cell population “workflow”. As shownin FIGS. 5C-E, targeting the enhancer region using either TALEN producedan Oct4-reduced stem cell population. The activity produced by thesingle-transcript TALEN was comparable to that of the traditional TALEN.

Example IV—Full Automation of Single-Transcript TALEN Assembly on aBiological Foundry

Many genomic studies may involve screening of a large number of targetswhich requires large-scale synthesis of TALENs to specifically disruptthese loci. Even though we have improved the efficiency and simplifiedthe workflow of TALEN synthesis, it is still very tedious if notimpossible to construct hundreds of these TALENs manually. Human errorsand inconsistency will also jeopardize the quality of the library.Automation has been used to accelerate biological organic engineering byeither reducing human interventions in individual steps, or completelyeliminating human intervention using integrated systems (Esvelt et al.,2011, “A system for the continuous directed evolution of biomolecules,”Nature, 472 (7344), p 499; Wang et al., 2009, “Programming cells bymultiplex genome organic engineering and accelerated evolution,” Nature,460 (7257), p 894). The latter approach has demonstrated the great powerof full automation by creating a large number of genetic variants in ashort time period. To enable large-scale applications of TALENs such asgenetic screening, we sought to fully automate the synthesis process“workflow” of TALENs. However, existing integrated platforms areextensively customized for specific tasks and difficult to reconfigure.It would not be efficient and economical to build a deeply customizedsystem dedicated to TALEN synthesis. Instead, we applied a generalizedGolden Gate assembly workflow implemented on iBioFAB.

The iBioFAB “system” consisted of component instruments, a centralrobotic platform, and a modular computational framework (FIG. 6). Twentydevices “instruments”, each in charge of a unit operation such aspipetting and incubation, were linked by two robotic arms “transporter”into various process modules such as DNA assembly and transformation,then further organized into workflows such as pathway construction andgenome organic engineering (FIGS. 6A-C). An overall scheduler“scheduler” was developed to orchestrate the unit operations and allowhierarchical programming of the workflows (FIG. 6C). The iBioFAB wasconfigured to perform a generalized automatic DNA assembly workflowwhere various kinds of DNA constructs “organic engineering targets” canbe manufactured on-demand with Golden Gate method (Engler et al., 2008,“A one pot, one step, precision cloning method with high throughputcapability,” PLoS One, 3 (11), e3647). A sequence of unit-operations wasdesigned to implement this workflow (FIG. 7A and FIG. 7B). To streamlinethe process, we developed Script Generator, a design tool thatautomatically converts DNA assembly “organic engineering targets”designs to experimental routines of mix-and-matching arbitrary DNA parts“unit operations”. Script Generator then generates robotic commands foriBioFAB to conduct the complex pipetting work “unit operation”. Thepipetting routes “unit operations and/or transport paths” were alsooptimized to minimize tip and time consumption. The aspiration steps“unit operations” are combined as much as possible for the samesubstrate and dispensed into corresponding destination. Tips are loadedon demand from the storage carousel “physical storage medium” to theliquid handling station.

In this work, we adapted this DNA assembly workflow for synthesizingsingle-transcript TALENs “organic engineering targets”. An extensionthat automated the DNA assembly design specifically for TALENs was addedto Script Generator. Using such pipeline, the operator only needs toinput the target DNA sequence “organic engineering target” to ScriptGenerator, and iBioFAB “system” would perform the rest of TALENsynthesis “workflow” with minimal human intervention. It only requiresthe operator “user” to load reagents and consumables on a daily basis.Any arbitrary number between 1 to 192 TALEN pairs can be synthesized ineach batch.

Example V—High-Throughput Synthesis of 192 Single-Transcript TALENs

To test the high-throughput synthesis pipeline “system”, we fed 192different human genomic target loci “organic engineering targets” toScript Generator “first device”. iBioFAB performed 3648 pipetting steps“from 444 different DNA parts and reagents within 17 hours at areasonable material cost. By staggering batches, over 400 TALENs can begenerated in a single day.

To evaluate the success rate of the synthesis, 94 randomly selectedconstructs “organic engineering targets” were verified by poly-clonalrestriction digestion “workflow”. All samples showed the correctdigestion pattern (FIG. 7C and FIG. 7D) which corresponds to a successrate of at least 96.2% with 95% confidence based on binomialprobability. For activity verification, we randomly selected 22 TALENsfor T7E1 assay in HEK293T cells (Mashal et al., 1995, “Detection ofmutations by cleavage of DNA heteroduplexes with bacteriophageresolvases,” Nat Genet, 9 (2), p 177). Here, 15 of 22 samples showedcleavage activity. Since cleavage activity was known to be sequencedependent, the lack of activity for some sites was not unexpected(Cermak et al., 2011, “Efficient design and assembly of custom TALEN andother TAL effector-based constructs for DNA targeting,” Nucleic AcidsRes, 39 (12), e82). To eliminate the possibility of mis-assembly, wesequenced all the constructs that did not show cleavage activity. Allsequencing reads aligned to the intended TALEN designs, indicating thatthe TALENs were correctly assembled (FIG. 12).

Besides TALEN, clustered regulatory short palindromic repeat(CRISPR)-Cas9 is another popular technology used in genome editingapplications (Sander et al., 2014, “CRISPR-Cas systems for editing,regulating and targeting genomes,” Nat Biotechnol, 32 (4), p 347). Asopposed to using a specific protein to recognize DNA sequences, CRISPRutilizes RNA to perform the recognition through base pairing. Using anucleic acid for targeting “organic engineering target” has manyadvantages, but most importantly, through the use of micro-array DNAsynthesis, a large nucleic acid library is readily accessible. As such,even though TALEN had a two-year head start over CRISPR, multipletargeting and genetic screening were both first achieved using CRISPR(Shalem et al., 2014, “Genome-scale CRISPR-Cas9 knockout screening inhuman cells,” Science, 343 (6166), p 84; Wang et al., 2014, “GeneticScreens in Human Cells Using the CRISPR-Cas9 System,” Science, 343(6166), p 80; Cong et al., 2013, “Multiplex Genome Engineering UsingCRISPR/Cas Systems,” Science, 339 (6121), p 819). However, due to itsrelatively short recognition sequence, 20 bp, off-target effect is asignificant problem in CRISPR (Fu et al., 2013, “High-frequencyoff-target mutagenesis induced by CRISPR-Cas nucleases in human cells,”Nat Biotechnol, 31 (9), p 822). In a genetic screening that targetsstructural genes, the off-target effect can be compensated by targetingmultiple sites within the same gene, so that high confidence hit can beidentified by looking for the enrichment of a set of sites instead ofany single site. However, in the case where the functional DNA elementis very small, e.g., a transcriptional enhancer, or a miRNA gene, thereis simply not enough length to fit in multiple targeting sites.Furthermore, in the case of an enhancer, the target cut sites aretranscription factor binding sites that are typically around 10 bp.Given the limited range for target selection, CRISPR may not be able tofind a site that is sufficiently unique in the genome. Furthermore,given the small number of selectable sites for such screens, the levelof confidence for any resultant hits will be low. A TALEN library, witha different off-target profile, can be used in conjunction with a CRISPRlibrary to improve the confidence of any potential hits.

In conclusion, we have developed a scheme to synthesize TALEN pairs on asingle vector in a one-pot reaction, which has substantially simplifiedthe synthesis of TALENs while achieving outstanding success rate. Anautomated process was developed accordingly, and the resulted pipelinemakes it possible to create large TALEN libraries at a reasonable costand time frame.

Methods—iBioFAB

iBioFAB “system” consists of a F5 robotic arm “transporter” (402) on a5-meter track “transport path”(Fanuc, Oshino-mura, Japan), an Evo200liquid handling robot “second transporter” (404) (Tecan, Männedorf,Switzerland), two shaking temperature controlled blocks (ThermoScientific, Waltham, Mass.), a M1000 microplate reader (406) (Tecan,Männedorf, Switzerland), a Cytomat 6000 incubator (408) (ThermoScientific, Waltham, Mass.), two Cytomat 2C shaking incubators (ThermoScientific, Waltham, Mass.), three Multidrop Combi reagent dispensers(412) (Thermo Scientific, Waltham, Mass.), four Trobot thermocyclers(414) (Biometra, Gottingen, Germany), Vspin plate centrifuge (Agilent,Santa Clara, Calif.), a storage carousel (416) (Thermo Scientific,Waltham, Mass.), a de-lidding station (Thermo Scientific, Waltham,Mass.), an Alps plate sealer (410) (Thermo Scientific, Waltham, Mass.),a WASP plate sealer (Thermo Scientific, Waltham, Mass.), a Xpeel sealpeeler (Brooks, Chelmsford, Mass.), and a label printer (418) (Agilent,USA). The liquid handling robot was equipped with an 8-channelindependent pipetter, a robotic manipulation arm, a 96-channel pipetter,six Peltier temperature controlled blocks (Torrey Pine, Carlsbad,Calif.), two shakers (Q.Instruments, Jena, Germany), a light box, and acamera for colony picking “a plurality of instruments” (Scirobotics,Kfar Saba, Israel), as partially shown in FIG. 2.

Momentum (Thermo Scientific, Waltham, Mass.) was used to communicatewith the peripheral devices, control the central robotic arm, andprogram process modules. Process modules defined the unit operations andsample transportation routes “transport path” between unit operations.Freedom Evoware (Tecan, Mannedorf, Switzerland) was used to control theliquid handling robot and program pipetting modules. Pipetting modulesspecifically defined the general procedure of pipetting on the liquidhandling robot, such as labware fetching “executable instruction” fromthe central robotic arm “transporter”, DNA part dispensing, reagentdispensing, and temperature controls “unit operations”. iScheduler andScriptGenerator are programed in Visual Basic. iScheduler executesprocess modules by sending commands in Extensible Markup Language toMomentum. The ScriptGenerator converted user defined DNA assembly“organic engineering target” as permutations of parts to source anddestination locations based on preloaded parts storage plate layouts.The corresponding pipetting routes “unit operations” were optimized byqueueing the destination locations from the same source. Pipettingworklists were compiled accordingly and sent to Freedom Evoware tocontrol aspiration, dispense, as well as tip change actions. Definedamount of each DNA part was aspirated and multi-dispensed withoutcontacting the liquid in the destination “executable instruction”. Tipswere re-used as much as possible and changed when all destinations forthe same source were dispensed. Constraints “interlocking condition”such as tip volume and maximum number of aspirations with each tip werealso imposed in the algorithm.

Example VI—Plasmids

Based on the RVDs parts used in the previous work (Liang et al., 2014,“FairyTALE: A high-throughput TAL effector synthesis platform,” ACSSynth Biol, 3 (2), p 67), a new library of TALEN stock plasmids weredeveloped for the single plasmid design. The group for position 6 wasreplaced with LR_N-term_FokI_P2A+C-term constructs (FIG. 8A). Dual andsingle RVD parts with NN were supplemented into the stock library. TheRVD and P2A fragments were inserted to a receiver plasmid (FIG. 8B) withhuman CMV promoter as well as last repeat, N terminus, and FokI domainfor the second TALEN monomer.

Example VII—Golden Gate Assembly and Verification

Golden Gate DNA assembly was performed with the methods described in theprevious work (Liang et al., 2014, “FairyTALE: A high-throughput TALeffector synthesis platform,” ACS Synth Biol, 3 (2), p 67). Competent E.coli HST08 strain (Clontech, Mountain View, Calif.) was prepared withMix & Go E. coli Transformation Buffer Set (Zymo Research, Irvine,Calif.). 2.5 μL of Golden Gate reaction products were first mixed withE. coli competent cells on a Peltier block held at 0° C. and incubatedfor 30 min. The cell plate was then transferred to a second Peltierblock held at 42° C. by the plate manipulation arm. After 1-min heatshock, the cell plate was transferred back to the 0° C. block andchilled for 2 min. The transformants were recovered in LB broth (Becton,Dickinson and Company, Franklin Lakes, N.J.) for 1 hr. The recoveredcell suspensions were either plated on LB agar media with 100 μg/mL ofampicillin or used to inoculate poly-clonally LB liquid mediasupplemented with 200 μg/mL of carbenicillin. Plasmids were extractedfrom the poly-clonal cultures with MagJET Plasmid DNA Kit (ThermoScientific, Waltham, Mass.) and restriction digested by EcoRI-HF (NewEngland Biolabs, Ipswich, Mass.). The digestion products were analyzedby 1% agarose gel in low throughput or Fragment Analyzer (AdvancedAnalytical Technologies, Ankeny, Iowa) in high throughput. Selectedplasmids were also verified by Sanger sequencing reactions (ACGT,Wheeling, Ill.) with 4 primers. The binomial probability confidenceinterval for assembly success rate was calculated with Clopper-Pearsonmethod (Clopper et al., 1934, “The use of confidence or fiducial limitsillustrated in the case of the binomial,” Biometrika, 26, p 404).

Example VIII—Mammalian Gene Knockout and Verification

Human embryonic kidney (HEK) cell line HEK293T was transfected withrandomly selected TALENs plasmids. HEK293T cells were used as they areeasy to cultivate and transfect. Although no cell authentication ormycoplasma contamination tests were performed, we reason that theresults of T7E1 assay is relatively insensitive to the cell linebackground. Cells were maintained in Dulbecco's modified Eagle's Medium(DMEM) (Corning Life Sciences, Tewksbury, Mass.) supplemented with 10%heat inactivated fetal bovine serum (Life Technologies, Carlsbad,Calif.) at 37° C. and 5% CO2 incubation. One day prior to transfection,293T cells were seeded into 12-well BioCoat Collagen-I coated plates(Corning Life Sciences, Tewksbury, Mass.) at a confluency of ˜50%.Transfections were performed with FuGENE HD Transfection Reagent(Promega, Madison, Wis.) according to the manufacturer's protocols.Briefly, for each well of the 12-well plate, 1 μg of clonally purifiedTALEN plasmid was first diluted in Opti-MEM (Life Technologies,Carlsbad, Calif.) to a total volume of 100 μL. After addition of 3 μLFugene HD reagent and incubation at room temperature for 5 min, the mixwas added onto the cells. Cells were harvested at 60 hourspost-transfection. The genomic DNA was extracted with QuickExtract DNAExtraction Solution (Epicentre, Madison, Wis.).

The cleavage efficiency was evaluated by T7E1 assay (Mashal et al.,1995, “Detection of mutations by cleavage of DNA heteroduplexes withbacteriophage resolvases,” Nat Genet, 9 (2), p 177). DNA amplicons weredesigned to have a length of 400-1000 bp flanking the nominal cleavagesite by a custom developed Visual Basic script. It searches the genomesequence within a given range for a pair of primer binding sites toavoid off-targets, long stretches of GC, AT, or any single type ofnucleotide. End nucleotides, GC contents, and melting temperatures wereoptimized. The relevant genome sequences were downloaded by queryingUCSC DAS server (www.genome.ucsc.edu/cgi-bin/das/) while off-targetcheck was performed by querying GGGenome server(www.gggenome.dbcls.jp/). The PCR amplification was conducted with Q5polymerase (New England Biolabs, Ipswich, Mass.) and annealingtemperature touchdown (65-55° C. for 10 cycles, 55° C. for 20 cycles).In the cleavage assay, 200 ng of purified amplicon in 10 μL NEB Buffer 2was first denatured and renatured (95° C., 5 min; 95-85° C. at −2° C./s;85-25° C. at −0.1° C./s; hold at 4° C.). 10U of T7 Endonuclease I (NewEngland Biolabs, Ipswich, Mass.) was added and incubated at 37° C. for15 min. The reaction was stopped by adding 1 μL of 0.5M EDTA. Thedigestion products were analyzed by Fragment Analyzer (AdvancedAnalytical Technologies, Ankeny, Iowa).

Example IX—Oct4 Down-Regulation Assay

TALEN constructs under evaluation were transfected into H1-Oct4-EGFPstem cells (WiCell, Madison, Wis.) by nucleofection according tomanufacturer's recommendations. After optimization, we settled on the P4Primary Cell 4D-Nucleofector Kit, and program CA-137 on the4D-Nucleofector (Lonza, Cologne, Germany). Cells were passaged one dayafter nucleofection, and were harvested on the fourth day afternucleofection. After harvest, the cells were counted and stained usingAlexa Fluor 647 conjugated SSEA4 antibody (Life Technologies, Carlsbad,Calif.) at a concentration of 5 x105 cells in 50 μL PBS with 2% BSA and2.5 μL SSEA4 antibody. The cells were stained in the dark at roomtemperature for 30 min, and washed 3 times in PBS before flow cytometryanalysis. During analysis, the stem cell population was first selectedby gating for the SSEA4 positive cells. Within this population, we thenlook at the spread of EGFP expression, and gate for the EGFP-reducedpopulation.

FIG. 2 illustrates an exemplary layout of iBioFAB's hardware. In anexemplary embodiment, iBioFAB has two robotic arms “transporters”. Acentralized 6-degree-of-freedom arm “transporter” on a 5-meter track isused to transport labware between instruments “transport path”. A3-degree-of-freedom arm “second transporter” moves labware inside theliquid handling station.

FIG. 4 illustrates a design and preliminary test of single-transcriptTALEN synthesis “organic engineering target”. FIG. 4A depicts theoverall design, wherein both TALENs were transcribed as one mRNA, butsliced to separate proteins in translation as a P2A sequence wasinserted between the open reading frames. FIG. 4B depicts the assemblyscheme. A library of all possible combinations of single and dual TALErepeats “organic engineering targets” were pre-assembled withstandardized Golden Gate linkers for each position. Thus, each TALENmonomer can target 8 to 15 nucleotides “organic engineering targets”with a mix of single and dual repeats. Repeats for both monomers as wellas the LR-C-terminus-Fok-I-P2A-N-terminus fragment are assembled in asingle Golden Gate assembly reaction. LR: last repeat. Term.: terminus.FIG. 4C illustrates a test assembly of a single-transcript TALEN pair.28 independent clones were picked and digested by PvuI and StuI. All hadcorrect digestion pattern. Arrows indicate the correct digestionpattern.

FIG. 5 illustrates a functional test of single-transcript TALENs. FIG.5A depicts single-transcript expression of a TALEN pair. Two distinctiveTALEN pairs were expressed in HEK293T cells with the single-transcriptdesign. TALEN monomers showed visible bands on Western blot while noband for the size of uncleaved doublet was detected. FIG. 5B depictsgenome editing in HEK293T cells. Single-transcript TALENs (STTLN) werecompared against the traditional dual plasmid TALENs (TDTLN) bytargeting BRCA2 as well as ABL1 sites in HEK293T cells. T7E1 assay wasperformed to detect the indel introduced by TALEN cleavage and NHEJ. TheSTTLN transfected samples showed comparable cleavage efficiency to TDTLNtransfected samples. CTRL: sample with no TALEN transfection served asnegative control. FIGS. 5C-E depict disruption of an Oct4 enhancer in H1hESC. Flow cytometry was used to quantify the GFP expression level inH1-Oct4-GFP cells. The gated population had lower than normal GFPexpression. Left: control population without enhancer disruption,middle: enhancer disrupted by traditional 2-plasmid TALEN, right:enhancer disrupted by single-transcript TALEN.

FIG. 6 depicts an overview of the iBioFAB system. FIG. 6A depicts abreakdown of unit operations according to an exemplary embodiment of thepresent disclosure. FIG. 6C illustrates an exemplary control hierarchyof iBioFAB. Process modules are developed in the system control GUI.iScheduler is in charge of workflow level control. Script Generatorgenerates pipetting routes for the liquid handling GUI. Process modulescan be quickly recombined to compose different workflows. Arrowsindicate flows of processes or samples. A user can choose to interveneat any time, such as moving a sample, instead of the transporter or canprocess samples, such as performing a unit operation, instead ofperipheral devices. Typically, samples or organic engineering targetsare processed in batches. Multiple batches can be scheduled andstaggered to be processed in parallel. In the programming interface, auser programs workflows with pre-developed and tested modules orsub-workflows. As previously described, the workflows define thedependency of unit operations for sample batches. The user designsbio-systems based on the organic engineering targets with help of aBioCAD. The designs are further converted to experimental plans byWorkflow Generator, which can integrate the ScriptGenerator therein. Insome embodiments, Workflow Generator only generates sample levelexperiment scripts that will be used as parameters and/or data by atransporter. In some embodiments, the Workflow Generator assistsprograming other unit operations or workflows. Nested sub-workflows,loops, and forks are allowed in workflows. These structures are thenlinearized for the scheduler. In case of large discrepancies between anactual runtime and schedule, or when triggered by a user, the workflowwill be rescheduled except for the steps to be executed immediately.Both actions and micro-steps or executable instructions are abstractedfor unit operations and defined in unit operation definitions. They arenot specific to any models of instruments. The drivers map micro-stepsto commands used in specific instruments.

FIG. 7 depicts a fully automated synthesis of TALEN libraries. FIG. 7Adepicts a general workflow for the DNA assembly pipeline based on GoldenGate method. Script generator converted project design ideas such aspermutations of DNA parts to assembly designs with appropriateextensions and further robotic commands for pipetting the stock plasmidsto DNA mixes. In Golden Gate reactions, Type IIs restriction enzymeslike BsaI generated a set of standard pre-characterized 4-bp singlestrained ends as linkers. The corresponding linkers annealed and wereligated by T4 ligase. FIG. 7B depicts a process flow diagram for thebuild step. Unit operations employed were marked in blue. FIG. 7C andFIG. 7D depict verification of single-transcript TALENs synthesized inhigh throughput. 94 samples were randomly selected from the 192 TALENpairs synthesized in the full batch test. Each plasmid sample encoding apair of TALENs was extracted from a polyclonal E. coli cell culture andrestriction digested. The fragment sizes were analyzed by capillaryelectrophoresis. The digestion pattern was simulated.

An exemplary summary of efficiency, throughput, and cost of such abiofoundry have shown to produce approximately 400 TALEN pairs per day,with approximately one hour of human labor required per day.

FIG. 8A and FIG. 8B depict a plasmid design for single plasmid TALENassembly. FIG. 8A depicts a P2A insert. It contained the last repeat,C-terminus, and FokI of the first TALEN monomer as well as theN-terminus of the second monomer. The two monomers were separated by aP2A sequence:

(SEQ ID. No.: 1) GGCAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGATGTGGAGGAGAACCCTGGACCTGGCATG.

FIG. 8B depicts a CMV receiver. The RVD inserts replaces ccdB fragmentduring Golden Gate reaction. The ccdB site was flanked by N-terminus ofthe first TALEN monomer and last repeat along with C-terminus and FokIdomain of the second monomer. A human CMV promoter is used to expressboth monomers. Four versions of the P2A insert as well as the CMVreceiver with different last repeats were constructed.

FIGS. 9A-H depict disrupting EGFP in HEK293 cells. AEF1a-tdTomato-P2A-EGFP cassette is stably expressed in HEK293T cells asa reporter system. TALENs are designed to cleave the EGFP fragment.tdTomato is used to exclude the non-expressive cells. FIG. 9A depictscells expressing tdTomato only. FIG. 9A depicts cells expressing bothtdTomato and EGFP, split by a P2A sequence. FIG. 9A depicts cellsexpressing EGFP only. FIG. 9A depicts negative control with TALENtargeting sequences other than EGFP. FIG. 9A depicts 2-plasmid TALENtargeting EGFP with NN for guanine. FIG. 9A depicts 2-plasmid TALENtargeting EGFP with NH for guanine. FIG. 9A depicts single-transcriptTALEN targeting EGFP with NN for guanine. FIG. 9A depictssingle-transcript TALEN targeting EGFP with NH for guanine. The resultsindicate that 1) single-transcript TALEN has comparable efficiency asthe 2-plasmid TALEN and 2) TALENs with NN has better efficiency than NH.

FIG. 11 depicts a list of substrates. The nucleotide target of each DBDis indicated with A, T, G, C, or D, denotes a RVD of NI, NG, NH, HD, orNN respectively. In the substrate plasmids, the DBD(s) are flanked byappropriate 4-bp junctions so that they can be assembled in appropriatepositions in the receiver plasmids by Golden Gate reaction. 5*substrates can be used to bridge Position 4 and 8 directly resulting ina shorter assembly if necessary. A, T, G, and C in P2A and Receiversubstrates denotes the targeted nucleotide by the last repeats ofTALENs. CMV indicates the CMV promoter used in this specific study whileother promoters can be used by supplementing new substrates to thelibrary. A yellow cell shows the substrates adapted from the previouswork (Liang et al., 2014, “FairyTALE: A high-throughput TAL effectorsynthesis platform,” ACS Synth Biol, 3 (2), p 67) while the rest ofsubstrates were supplemented in this study.

FIG. 12 depicts a list of results from T7E1 assay. HEK293T cells weretransfected with TALENs targeting 22 randomly selected genomic loci.Genomic DNA samples extracted from polyclonal post-transfection cultureswere used for T7E1 assay. Asterisks “*” denote TALENs showing nocleavage activity were DNA sequencing-verified and aligned with thedesign.

REFERENCES CITED AND ALTERNATIVE EMBODIMENTS

For convenience in explanation and accurate definition in the appendedclaims, the terms “upper”, “lower”, “up”, “down”, “upwards”,“downwards”, “inner”, “outer”, “inside”, “outside”, “inwardly”,“outwardly”, “interior”, “exterior”, “front”, “rear”, “back”,“forwards”, and “backwards” are used to describe features of theexemplary embodiments with reference to the positions of such featuresas displayed in the figures.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The present invention can be implemented as a computer program productthat comprises a computer program mechanism embedded in a nontransitorycomputer readable storage medium. For instance, the computer programproduct could contain the program modules shown in any combination ofFIG. 1 or 2 and/or described in FIG. 3. These program modules can bestored on a CD-ROM, DVD, magnetic disk storage product, USB key, or anyother non-transitory computer readable data or program storage product.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical applications, to thereby enable others skilled in the artto best utilize the invention and various embodiments with variousmodifications as are suited to the particular use contemplated. Theinvention is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled.

What is claimed is:
 1. A method of implementing one or more workflows:at a device comprising one or more processors, memory storing one ormore programs for execution by the one or more processors, a controller,a transport path, a communications interface in electrical communicationwith at least the transport path, a power supply, a plurality ofperipheral devices that includes a transporter and an articulatedhandling robot attached to the transporter, wherein the transporter isconfigured to autonomously move about the transport path and thearticulated handling robot is configured to move a first tray between atleast a first instrument and a second instrument in a plurality ofinstruments; the one or more programs singularly or collectively usingthe one or more processors to execute a method comprising: (A)obtaining, in electronic form, a first plurality of organic engineeringtargets; (B) assigning, via the controller, the first plurality oforganic engineering targets to a first uncompiled workflow, wherein thefirst uncompiled workflow is configured to produce the first pluralityof organic engineering targets, the first uncompiled workflow isassociated with a first subset of process modules in a plurality ofprocess modules, each respective process module in the plurality ofprocess modules is associated with a different subset of unit operationdefinitions in a plurality of unit operation definitions, eachrespective unit operation definition in the plurality of unit operationdefinitions is independently associated with a corresponding timeinterval, and each respective unit operation definition in the pluralityof unit operations is independently associated with a first subset ofinstruments in the plurality of instruments; (C) translating, via thecontroller, for each respective organic engineering target in the firstplurality of organic engineering targets, the first uncompiled workflowinto a corresponding instance of a compiled first workflow for therespective organic engineering target, wherein the correspondinginstance of the compiled first workflow comprises: (i) for eachrespective instrument in the first subset of instruments: (a) an addressof the respective instrument comprising: a coarse grain address of therespective instrument that is associated with a marker of the respectiveinstrument, and (b) one or more execution instructions comprising: acoarse traverse instruction that commands the transporter to traverse tothe respective marker of the respective instrument, and a fine traverseinstruction that commands the articulated handling robot to move to atleast one spatial coordinate associated with the respective instrument,(ii) a first plurality of unit operations, wherein the first pluralityof unit operations is temporally organized into a linear temporal order,and wherein each respective unit operation in the first plurality ofunit operations is characterized by the time interval of thecorresponding unit operation definition, thereby forming a plurality ofinstances of the compiled first workflow.
 2. The method of claim 1,wherein the transporter further comprises a positioning system that isconfigured to locate and guide the transporter within the transportpath.
 3. The method of claim 2, wherein: the positioning systemcomprises a global positioning system, and the respective marker of therespective instrument is a global positioning system coordinate.
 4. Themethod of claim 2, wherein the positioning system comprises arepresentation of a marker positioning of the transport path.
 5. Themethod of claim 4, wherein the representation of the marker positioningof the transport path is either an image feed or a video feed.
 6. Themethod of claim 5, wherein either a low-pass filter or a Gaussianblurring function is applied to the representation of the markerpositioning of the transport path.
 7. The method of claim 2, wherein thetransporter further comprises an inertial measurement unit that isconfigured to verify and correct each execution of the coarse traverseinstruction of the respective instruments.
 8. The method of claim 2,wherein the articulated handling robot further comprises a plurality ofsensors, wherein each sensor in the plurality of sensors is configuredto provide information related to a spatial location of the articulatedhandling robot and a marker positioning of each visible marker.
 9. Themethod of claim 8, wherein the transporter further comprises aproportional-integral-derivative controller that is in electricalcommunication with the plurality of sensors in order to maintain aspatial location of the transporter.
 10. The method of claim 8, wherein:the articulated handling robot is configured to transfer the first trayto a first instrument in the plurality of instruments using a safetytransfer procedure when a readout from the plurality of sensors fails tosatisfy a threshold proximity value with respect to an actual locationof the first instrument; and the articulated handling robot isconfigured to transfer the first tray to the first instrument using adefault transfer procedure when the readout from the plurality ofsensors satisfies the threshold proximity value with respect to theactual physical location of the first instrument.
 11. A system forimplementing workflows comprising a first device, wherein the firstdevice comprises: a display; a power supply; a communications interface;one or more peripheral devices including a reagent handling device, thereagent handling device comprising: a wash manifold, a primary valvethat regulates an internal flow path in the reagent handling device, aplurality of reagent containers, wherein each reagent container in theplurality of reagent containers comprises a valve that controls a flowfrom the respective reagent container to the primary valve, asterilization container configured to hold a sterilizing fluid, thesterilization container comprising a valve that regulates a flow of thesterilization fluid either to the primary valve or to the wash manifold,the wash manifold is coupled to the respective valve of each reagentcontainer in the plurality of reagent containers, wherein the washmanifold regulates the flow of the sterilization fluid from thesterilization container to the plurality of reagent containers, and adispense manifold that is coupled to the primary valve and comprises aplurality of dispensers, the dispense manifold configured to control aflow from the primary valve to the plurality of dispensers; one or moreprocessors; memory; and one or more programs, wherein the one or moreprograms are stored in the memory and are configured to be executed bythe one or more processors, the one or more programs includinginstructions for: (A) obtaining, in electronic form, a first pluralityof organic engineering targets; (B) assigning the first plurality oforganic engineering targets to a first uncompiled workflow, wherein thefirst uncompiled workflow is configured to produce the first pluralityof organic engineering targets, the first uncompiled workflow isassociated with a first subset of process modules in a plurality ofprocess modules, each respective process module in the plurality ofprocess modules is associated with a different subset of unit operationdefinitions in a plurality of unit operation definitions, eachrespective unit operation definition in the plurality of unit operationdefinitions is independently associated with a corresponding timeinterval, and each respective unit operation definition in the pluralityof unit operations is independently associated with a first subset ofinstruments in a plurality of instruments; (C) translating, for eachrespective organic engineering target in the first plurality of organicengineering targets, the first uncompiled workflow into a correspondinginstance of a compiled first workflow for the respective organicengineering target, wherein the corresponding instance of the compiledfirst workflow comprises: (i) for each respective instrument in thefirst subset of instruments (a) an address of the respective instrumentand (b) one or more execution instructions for the respectiveinstrument, and (ii) a first plurality of unit operations, wherein thefirst plurality of unit operations is temporally organized into a lineartemporal order, and wherein, each respective unit operation in the firstplurality of unit operations is characterized by the time interval ofthe corresponding unit operation definition, thereby forming a pluralityof instances of the compiled first workflow.
 12. The system of claim 11,wherein a pressure regulator is coupled to each of the sterilizationcontainer, the primary valve, and each reagent container in theplurality of reagent containers.
 13. The system of claim 12, whereineach pressure regulator further comprises a filter that is configured toprevent contamination of the respective reagent container.
 14. Thesystem of claim 12, wherein each pressure regulator further comprises acheck valve.
 15. The system of claim 11, wherein the reagent handlingdevice further comprises a pump coupled to the primary valve that drivesthe internal flow path of the reagent handling device.
 16. The system ofclaim 11, wherein the reagent handling device further comprises adispensing cycle that is configured to dispense a selection from apredetermined set of reagent containers in the plurality of reagentcontainers.
 17. The system of claim 11, wherein the reagent handlingdevice further comprises a first sterilization cycle that is configuredto sterilize a portion of the system.
 18. The system of claim 11,wherein the reagent handling device further comprises a secondsterilization cycle that is configured to purge a portion of the system.19. The system of claim 11, wherein each component of the reagenthandling device is coupled through removable tubing.
 20. The system ofclaim 11, wherein human intervention is only required to replenish arespective reagent of each reagent container in the plurality of reagentcontainers and to replenish the sterilization fluid of the sterilizationcontainer.
 21. A method of implementing one or more workflows: at adevice comprising one or more processors, memory storing one or moreprograms for execution by the one or more processors, a controller, aplurality of instruments including a multi-well plate centrifugeinstrument, a communications interface in electrical communication withat least the plurality of instruments, and a power supply; the one ormore programs singularly or collectively using the one or moreprocessors to execute a method comprising: (A) obtaining in electronicform, a first plurality of organic engineering targets; (B) assigning,via the controller, the first plurality of organic engineering targetsto a first uncompiled workflow, wherein the first uncompiled workflow isconfigured to produce each organic engineering target in the firstplurality of organic engineering targets in a corresponding well in aplate in a first set of multi-well plates, the first uncompiled workflowis associated with a first subset of process modules in a plurality ofprocess modules, each respective process module in the plurality ofprocess modules is associated with a different subset of unit operationdefinitions in a plurality of unit operation definitions, eachrespective unit operation definition in the plurality of unit operationdefinitions is independently associated with a corresponding timeinterval, and each respective unit operation definition in the pluralityof unit operations is independently associated with a first subset ofinstruments in the plurality of instruments, wherein the first subset ofinstruments includes the multi-well plate centrifuge instrument; and (C)translating, via the controller, for each respective organic engineeringtarget in the first plurality of organic engineering targets, the firstuncompiled workflow into a corresponding instance of a compiled firstworkflow for the respective organic engineering target, wherein thecorresponding instance of the compiled first workflow comprises: (i) foreach respective instrument in the first subset of instruments (a) anaddress of the respective instrument, and (b) one or more executioninstructions, wherein the one or more execution instructions for amulti-well plate centrifuge instrument comprise: b.i) determining a massof each multi-well plate in a first set of multi-well plates, b.ii)disposing each multi-well plate in the first set of multi-well platesinto the multi-well plate centrifuge, wherein each respective multi-wellplate in the first set of multi-well plates has a corresponding counterbalance in a set of counter balances disposed in the multi-well platecentrifuge at a position opposite the respective multi-well plate,b.iii) adjusting, without human intervention, a mass of each respectivecounter balance in the set of counter balances to be equal to thecorresponding multi-well plate in the first set of multi-well plates,and b.iv) operating the multi-well centrifuge, and (ii) a firstplurality of unit operations, wherein the first plurality of unitoperations is temporally organized into a linear temporal order, andwherein each respective unit operation in the first plurality of unitoperations is characterized by the time interval of the correspondingunit operation definition, thereby forming a plurality of instances ofthe compiled first workflow.
 22. The method of claim 21, wherein thefirst set of multi-well plates is a set of one multi-well plate.
 23. Themethod of claim 21, wherein the first set of multi-well plates is a setof from two multi-well plates to five multi-well plates.
 24. The methodof claim 21, wherein the adjusting b.iii) further comprises pumpingfluid to the respective counter balance in order to adjust the masstherein.
 25. The method of claim 21, wherein the adjusting b.iii)further comprises drawing fluid from the respective counter balance inorder to adjust the mass therein.
 26. The method of claim 21, whereineach counter balance in the set of counter balances comprises a bottomend portion that is configured to pool fluid therein.
 27. The method ofclaim 21, wherein the determining b.i) and the adjusting b.iii) areperformed simultaneously.
 28. The method of claim 21, wherein theadjusting b.iii) further comprises storing the adjusted mass of eachrespective counter balance in the set of counter balances.
 29. Themethod of claim 21, wherein the one or more instructions for themulti-well plate centrifuge instrument further comprises: b.v)determining a mass of each multi-well plate in a second set ofmulti-well plates, b.vi) disposing each multi-well plate in the secondset of multi-well plates into the centrifuge, wherein each respectivemulti-well plate in the second set of multi-well plates has acorresponding counter balance in a second set of counter balancesdisposed in the multi-well plate centrifuge at a position opposite therespective multi-well plate, b.vii) adjusting, without humanintervention, the mass of each counter balance in the second set ofcounter balances to be equal to the mass of the corresponding multi-wellplate in the second set of multi-well plates, and b.viii) operating themulti-well plate centrifuge.
 30. The method of claim 29, wherein thesecond set of multi-well plates is the first set of multi-well plates.